Dosages of immunoconjugates of antibodies and sn-38 for improved efficacy and decreased toxicity

ABSTRACT

The present invention relates to therapeutic immunoconjugates comprising SN-38 attached to an anti-Trop-2 antibody or antigen-binding antibody fragment. In preferred embodiments, the antibody may be an hRS7 antibody. The methods and compostions are of use to treat Trop-2 expressing cancers in human patients, preferably in patients who are resistant to or relapsed from at least one prior anti-cancer therapy, more preferably in patients who are resistant to or relapsed from treatment with irinotecan. The immunoconjugate may be administered at a dosage of 3 mg/kg to 18 mg/kg, preferably 8 to 12 mg/kg, more preferably 8 to 10 mg/kg. When administered at specified dosages and schedules, the immunoconjugate can reduce solid tumors in size and reduce or eliminate metastases. Preferred tumors to treat with the subject immunoconjugates include triple-negative breast cancer, HER+, ER+, progesterone+ breast cancer, metastatic non-small-cell lung cancer, a metastatic small-cell lung cancer and metastatic pancreatic cancer.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.16/155,423, filed Oct. 9, 2018, which was a divisional of U.S. patentapplication Ser. No. 15/069,208 (now U.S. Pat. No. 10,137,196), filedMar. 14, 2016, which was a continuation-in-part of U.S. patentapplication Ser. No. 14/667,982 (now U.S. Pat. No. 9,493,573), filedMar. 25, 2015, which was a divisional of U.S. patent application Ser.No. 13/948,732 (now U.S. Pat. No. 9,028,833), filed Jul. 23, 2013, whichclaimed the benefit under 35 U.S.C. 119(e) of U.S. Provisional PatentApplications 61/736,684, filed Dec. 13, 2012, and 61/749,548, filed Jan.7, 2013. This application claims the benefit under 35 U.S.C. 119(e) ofU.S. Provisional Patent Applications 62/133,654, filed Mar. 16, 2015,62/133,729, filed Mar. 16, 2015, 62/138,092, filed Mar. 25, 2015,62/156,608, filed May 4, 2015, and 62/241,881, filed Oct. 15, 2015, thetext of each priority application incorporated herein by reference inits entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under grant 1 R43CA171388 awarded by the National Institutes of Health. The U.S.government has certain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Mar. 8, 2016, isnamed IMM356US1 SL.txt and is 26,230 bytes in size.

FIELD OF THE INVENTION

The present invention relates to therapeutic use of immunoconjugates ofantibodies or antigen-binding antibody fragments and camptothecins, suchas SN-38, with improved ability to target various cancer cells in humansubjects. In preferred embodiments, the antibodies and therapeuticmoieties are linked via an intracellularly-cleavable linkage thatincreases therapeutic efficacy. In more preferred embodiments, theimmunoconjugates are administered at specific dosages and/or specificschedules of administration that optimize the therapeutic effect. Theoptimized dosages and schedules of administration of SN-38-conjugatedantibodies for human therapeutic use disclosed herein show unexpectedsuperior efficacy that could not have been predicted from animal modelstudies, allowing effective treatment of cancers that are resistant tostandard anti-cancer therapies, including irinotecan (CPT-11), theparent compound of SN-38. Most preferably, the methods and compositionsare of use to treat Trop-2 positive cancer, using an anti-Trop-2hRS7-SN-38 immunoconjugate. In specific embodiments, the immunoconjugatemay be administered to a human subject with a Trop-2 positive cancer ata dosage of between 3 and 18 mg/kg, more preferably between 4 and 12mg/kg, most preferably between 8 and 10 mg/kg. In other preferredembodiments, the methods and compositions may be used to treat Trop-2positive cancer that is relapsed from or refractory to other standardanti-cancer therapies. Surprisingly, the anti-Trop-2-SN38 antibody drugconjugates (ADCs) are effective to treat Trop-2 positive cancers inpatients who had relapsed from or shown resistance to treatmentscomprising irinotecan therapy, such as pancreatic cancer,triple-negative breast cancer, small cell lung cancer and non-small celllung cancer.

BACKGROUND OF THE INVENTION

For many years it has been an aim of scientists in the field ofspecifically targeted drug therapy to use monoclonal antibodies (MAbs)for the specific delivery of toxic agents to human cancers. Conjugatesof tumor-associated MAbs and suitable toxic agents have been developed,but have had mixed success in the therapy of cancer in humans, andvirtually no application in other diseases, such as infectious andautoimmune diseases. The toxic agent is most commonly a chemotherapeuticdrug, although particle-emitting radionuclides, or bacterial or planttoxins, have also been conjugated to MAbs, especially for the therapy ofcancer (Sharkey and Goldenberg, C A Cancer J Clin. 2006 July-August;56(4):226-243) and, more recently, with radioimmunoconjugates for thepreclinical therapy of certain infectious diseases (Dadachova andCasadevall, Q J Nucl Med Mol Imaging 2006; 50(3):193-204).

The advantages of using MAb-chemotherapeutic drug conjugates are that(a) the chemotherapeutic drug itself is structurally well defined; (b)the chemotherapeutic drug is linked to the MAb protein using verywell-defined conjugation chemistries, often at specific sites remotefrom the MAbs' antigen binding regions; (c) MAb-chemotherapeutic drugconjugates can be made more reproducibly and usually with lessimmunogenicity than chemical conjugates involving MAbs and bacterial orplant toxins, and as such are more amenable to commercial developmentand regulatory approval; and (d) the MAb-chemotherapeutic drugconjugates are orders of magnitude less toxic systemically thanradionuclide MAb conjugates, particularly to the radiation-sensitivebone marrow.

Camptothecin (CPT) and its derivatives are a class of potent antitumoragents. Irinotecan (also referred to as CPT-11) and topotecan are CPTanalogs that are approved cancer therapeutics (Iyer and Ratain, CancerChemother. Phamacol. 42: S31-S43 (1998)). CPTs act by inhibitingtopoisomerase I enzyme by stabilizing topoisomerase I-DNA complex (Liu,et al. in The Camptothecins: Unfolding Their Anticancer Potential, LiehrJ. G., Giovanella, B. C. and Verschraegen (eds), NY Acad Sci., NY922:1-10 (2000)). CPTs present specific issues in the preparation ofconjugates. One issue is the insolubility of most CPT derivatives inaqueous buffers. Second, CPTs provide specific challenges for structuralmodification for conjugating to macromolecules. For instance, CPT itselfcontains only a tertiary hydroxyl group in ring-E. The hydroxylfunctional group in the case of CPT must be coupled to a linker suitablefor subsequent protein conjugation; and in potent CPT derivatives, suchas SN-38, the active metabolite of the chemotherapeutic CPT-11, andother C-10-hydroxyl-containing derivatives such as topotecan and10-hydroxy-CPT, the presence of a phenolic hydroxyl at the C-10 positioncomplicates the necessary C-20-hydroxyl derivatization. Third, thelability under physiological conditions of the δ-lactone moiety of theE-ring of camptothecins results in greatly reduced antitumor potency.Therefore, the conjugation protocol is performed such that it is carriedout at a pH of 7 or lower to avoid the lactone ring opening. However,conjugation of a bifunctional CPT possessing an amine-reactive groupsuch as an active ester would typically require a pH of 8 or greater.Fourth, an intracellularly-cleavable moiety preferably is incorporatedin the linker/spacer connecting the CPTs and the antibodies or otherbinding moieties.

A need exists for more effective methods of preparing and administeringantibody-CPT conjugates, such as antibody-SN-38 conjugates. Preferably,the methods comprise optimized dosing and administration schedules thatmaximize efficacy and minimize toxicity of the antibody-CPT conjugatesfor therapeutic use in human patients.

SUMMARY OF THE INVENTION

As used herein, the abbreviation “CPT” may refer to camptothecin or anyof its derivatives, such as SN-38, unless expressly stated otherwise.The present invention resolves an unfulfilled need in the art byproviding improved methods and compositions for preparing andadministering CPT-antibody immunoconjugates. Preferably, thecamptothecin is SN-38. The disclosed methods and compositions are of usefor the treatment of a variety of diseases and conditions which arerefractory or less responsive to other forms of therapy, and can includediseases against which suitable antibodies or antigen-binding antibodyfragments for selective targeting can be developed, or are available orknown. Preferred diseases or conditions that may be treated with thesubject immunoconjugates include, for example, Trop-2 positive cancer.

Preferably, the targeting moiety is an antibody, antibody fragment,bispecific or other multivalent antibody, or other antibody-basedmolecule or compound. The antibody can be of various isotypes,preferably human IgG1, IgG2, IgG3 or IgG4, more preferably comprisinghuman IgG1 hinge and constant region sequences. The antibody or fragmentthereof can be a chimeric human-mouse, a chimeric human-primate, ahumanized (human framework and murine hypervariable (CDR) regions), orfully human antibody, as well as variations thereof, such as half-IgG4antibodies (referred to as “unibodies”), as described by van der NeutKolfschoten et al. (Science 2007; 317:1554-1557). More preferably, theantibody or fragment thereof may be designed or selected to comprisehuman constant region sequences that belong to specific allotypes, whichmay result in reduced immunogenicity when the immunoconjugate isadministered to a human subject. Preferred allotypes for administrationinclude a non-G1m1 allotype (nG1m1), such as G1m3, G1m3,1, G1m3,2 orG1m3,1,2. More preferably, the allotype is selected from the groupconsisting of the nG1m1, G1m3, nG1m1,2 and Km3 allotypes.

Antibodies of use may bind to any disease-associated antigen known inthe art. Where the disease state is cancer, for example, many antigensexpressed by or otherwise associated with tumor cells are known in theart, including but not limited to, carbonic anhydrase IX,alpha-fetoprotein (AFP), α-actinin-4, A3, antigen specific for A33antibody, ART-4, B7, Ba 733, BAGE, BrE3-antigen, CA125, CAMEL, CAP-1,CASP-8/m, CCL19, CCL21, CD1, CD1a, CD2, CD3, CD4, CD5, CD8, CD11A, CD14,CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD29, CD30, CD32b,CD33, CD37, CD38, CD40, CD40L, CD44, CD45, CD46, CD52, CD54, CD55, CD59,CD64, CD66a-e, CD67, CD70, CD70L, CD74, CD79a, CD80, CD83, CD95, CD126,CD132, CD133, CD138, CD147, CD154, CDCl₂7, CDK-4/m, CDKN2A, CTLA-4,CXCR4, CXCR7, CXCL12, HIF-1a, colon-specific antigen-p (CSAp), CEA(CEACAM5), CEACAM6, c-Met, DAM, EGFR, EGFRvIII, EGP-1 (TROP-2), EGP-2,ELF2-M, Ep-CAM, fibroblast growth factor (FGF), Flt-1, Flt-3, folatereceptor, G250 antigen, GAGE, gp100, GRO-(3, HLA-DR, HM1.24, humanchorionic gonadotropin (HCG) and its subunits, HER2/neu, HMGB-1, hypoxiainducible factor (HIF-1), HSP70-2M, HST-2, Ia, IGF-1R, IFN-γ, IFN-α,IFN-β, IFN-λ, IL-4R, IL-6R, IL-13R, IL-15R, IL-17R, IL-18R, IL-2, IL-6,IL-8, IL-12, IL-15, IL-17, IL-18, IL-23, IL-25, insulin-like growthfactor-1 (IGF-1), KC4-antigen, KS-1-antigen, KS1-4, Le-Y, LDR/FUT,macrophage migration inhibitory factor (MIF), MAGE, MAGE-3, MART-1,MART-2, NY-ESO-1, TRAG-3, mCRP, MCP-1, MIP-1A, MIP-1B, MIF, MUC1, MUC2,MUC3, MUC4, MUC5ac, MUC13, MUC16, MUM-1/2, MUM-3, NCA66, NCA95, NCA90,PAM4 antigen, pancreatic cancer mucin, PD-1 receptor, placental growthfactor, p53, PLAGL2, prostatic acid phosphatase, PSA, PRAME, PSMA, P1GF,ILGF, ILGF-1R, IL-6, IL-25, RS5, RANTES, T101, SAGE, 5100, survivin,survivin-2B, TAC, TAG-72, tenascin, TRAIL receptors, TNF-α, Tn antigen,Thomson-Friedenreich antigens, tumor necrosis antigens, VEGFR, ED-Bfibronectin, WT-1, 17-1A-antigen, complement factors C3, C3a, C3b, C5a,C5, an angiogenesis marker, bcl-2, bcl-6, Kras, an oncogene marker andan oncogene product (see, e.g., Sensi et al., Clin Cancer Res 2006,12:5023-32; Parmiani et al., J Immunol 2007, 178:1975-79; Novellino etal. Cancer Immunol Immunother 2005, 54:187-207). Preferably, theantibody binds to CEACAM5, CEACAM6, EGP-1 (TROP-2), MUC-16, AFP,MUC5a,c, PAM4 antigen, CD74, CD19, CD20, CD22 or HLA-DR. Mostpreferably, the antibody binds to Trop-2.

Exemplary antibodies that may be utilized include, but are not limitedto, hR1 (anti-IGF-1R, U.S. patent application Ser. No. 12/722,645, filedMar. 12, 2010), hPAM4 (anti-mucin, U.S. Pat. No. 7,282,567), hA20(anti-CD20, U.S. Pat. No. 7,251,164), hA19 (anti-CD19, U.S. Pat. No.7,109,304), hIMMU31 (anti-AFP, U.S. Pat. No. 7,300,655), hLL1(anti-CD74, U.S. Pat. No. 7,312,318), hLL2 (anti-CD22, U.S. Pat. No.7,074,403), hMu-9 (anti-CSAp, U.S. Pat. No. 7,387,773), hL243(anti-HLA-DR, U.S. Pat. No. 7,612,180), hMN-14 (anti-CEACAM5, U.S. Pat.No. 6,676,924), hMN-15 (anti-CEACAM6, U.S. Pat. No. 7,541,440), hRS7(anti-EGP-1, U.S. Pat. No. 7,238,785), hMN-3 (anti-CEACAM6, U.S. Pat.No. 7,541,440), Ab124 and Ab125 (anti-CXCR4, U.S. Pat. No. 7,138,496),the Examples section of each cited patent or application incorporatedherein by reference. More preferably, the antibody is IMMU-31(anti-AFP), hRS7 (anti-TROP-2), hMN-14 (anti-CEACAM5), hMN-3(anti-CEACAM6), hMN-15 (anti-CEACAM6), hLL1 (anti-CD74), hLL2(anti-CD22), hL243 or IMMU-114 (anti-HLA-DR), hA19 (anti-CD19) or hA20(anti-CD20). As used herein, the terms epratuzumab and hLL2 areinterchangeable, as are the terms veltuzumab and hA20, hL243g4P,hL243gamma4P and IMMU-114.

Alternative antibodies of use include, but are not limited to, abciximab(anti-glycoprotein IIb/IIIa), alemtuzumab (anti-CD52), bevacizumab(anti-VEGF), cetuximab (anti-EGFR), gemtuzumab (anti-CD33), ibritumomab(anti-CD20), panitumumab (anti-EGFR), rituximab (anti-CD20), tositumomab(anti-CD20), trastuzumab (anti-ErbB2), lambrolizumab (anti-PD-1receptor), nivolumab (anti-PD-1 receptor), ipilimumab (anti-CTLA-4),abagovomab (anti-CA-125), adecatumumab (anti-EpCAM), atlizumab(anti-IL-6 receptor), benralizumab (anti-CD125), obinutuzumab (GA101,anti-CD20), CC49 (anti-TAG-72), AB-PG1-XG1-026 (anti-PSMA, U.S. patentapplication Ser. No. 11/983,372, deposited as ATCC PTA-4405 andPTA-4406), D2/B (anti-PSMA, WO 2009/130575), tocilizumab (anti-IL-6receptor), basiliximab (anti-CD25), daclizumab (anti-CD25), efalizumab(anti-CD11a), GA101 (anti-CD20; Glycart Roche), muromonab-CD3 (anti-CD3receptor), natalizumab (anti-a4 integrin), omalizumab (anti-IgE);anti-TNF-α antibodies such as CDP571 (Ofei et al., 2011, Diabetes45:881-85), MTNFAI, M2TNFAI, M3TNFAI, M3TNFABI, M302B, M303 (ThermoScientific, Rockford, Ill.), infliximab (Centocor, Malvern, Pa.),certolizumab pegol (UCB, Brussels, Belgium), anti-CD40L (UCB, Brussels,Belgium), adalimumab (Abbott, Abbott Park, Ill.), Benlysta (Human GenomeSciences); antibodies for therapy of Alzheimer's disease such as Alz 50(Ksiezak-Reding et al., 1987, J Biol Chem 263:7943-47), gantenerumab,solanezumab and infliximab; anti-fibrin antibodies like 59D8, T2G1s,MH1; anti-CD38 antibodies such as MOR03087 (MorphoSys AG), MOR202(Celgene), HuMax-CD38 (Genmab) or daratumumab (Johnson & Johnson).

In a preferred embodiment, the chemotherapeutic moiety is selected fromcamptothecin (CPT) and its analogs and derivatives and is morepreferably SN-38. However, other chemotherapeutic moieties that may beutilized include taxanes (e.g, baccatin III, taxol), epothilones,anthracyclines (e.g., doxorubicin (DOX), epirubicin,morpholinodoxorubicin (morpholino-DOX), cyanomorpholino-doxorubicin(cyanomorpholino-DOX), 2-pyrrolinodoxorubicin (2-PDOX) or a prodrug formof 2-PDOX (pro-2-PDOX); see, e.g., Priebe W (ed.), ACS symposium series574, published by American Chemical Society, Washington D.C., 1995(332pp) and Nagy et al., Proc. Natl. Acad. Sci. USA 93:2464-2469, 1996),benzoquinoid ansamycins exemplified by geldanamycin (DeBoer et al.,Journal of Antibiotics 23:442-447, 1970; Neckers et al., Invest. NewDrugs 17:361-373, 1999), and the like. Preferably, the antibody orfragment thereof links to at least one chemotherapeutic moiety;preferably 1 to about 5 chemotherapeutic moieties; more preferably 6 ormore chemotherapeutic moieties, most preferably about 6 to about 12chemotherapeutic moieties.

An example of a water soluble CPT derivative is CPT-11. Extensiveclinical data are available concerning CPT-11's pharmacology and its invivo conversion to the active SN-38 (Iyer and Ratain, Cancer ChemotherPharmacol. 42:S31-43 (1998); Mathijssen et al., Clin Cancer Res.7:2182-2194 (2002); Rivory, Ann NY Acad Sci. 922:205-215, 2000)). Theactive form SN-38 is about 2 to 3 orders of magnitude more potent thanCPT-11. In specific preferred embodiments, the immunoconjugate may be anhMN-14-SN-38, hMN-3-SN-38, hMN-15-SN-38, IMMU-31-SN-38, hRS7-SN-38,hA20-SN-38, hL243-SN-38, hLL1-SN-38 or hLL2-SN-38 conjugate.

Various embodiments may concern use of the subject methods andcompositions to treat a cancer, including but not limited tonon-Hodgkin's lymphomas, B-cell acute and chronic lymphoid leukemias,Burkitt lymphoma, Hodgkin's lymphoma, acute large B-cell lymphoma, hairycell leukemia, acute myeloid leukemia, chronic myeloid leukemia, acutelymphocytic leukemia, chronic lymphocytic leukemia, T-cell lymphomas andleukemias, multiple myeloma, Waldenstrom's macroglobulinemia,carcinomas, melanomas, sarcomas, gliomas, bone, and skin cancers. Thecarcinomas may include carcinomas of the oral cavity, esophagus,gastrointestinal tract, pulmonary tract, lung, stomach, colon, breast,ovary, prostate, uterus, endometrium, cervix, urinary bladder, pancreas,bone, brain, connective tissue, liver, gall bladder, urinary bladder,kidney, skin, central nervous system and testes. Preferably, the cancerexpresses Trop-2 antigen.

In certain embodiments involving treatment of cancer, the drugconjugates may be used in combination with surgery, radiation therapy,chemotherapy, immunotherapy with naked antibodies, radioimmunotherapy,immunomodulators, vaccines, and the like. These combination therapiescan allow lower doses of each therapeutic to be given in suchcombinations, thus reducing certain severe side effects, and potentiallyreducing the courses of therapy required. When there is no or minimaloverlapping toxicity, ful doses of each can also be given.

Preferred optimal dosing of immunoconjugates may include a dosage ofbetween 3 mg/kg and 18 mg/kg, preferably given either weekly, twiceweekly or every other week. The optimal dosing schedule may includetreatment cycles of two consecutive weeks of therapy followed by one,two, three or four weeks of rest, or alternating weeks of therapy andrest, or one week of therapy followed by two, three or four weeks ofrest, or three weeks of therapy followed by one, two, three or fourweeks of rest, or four weeks of therapy followed by one, two, three orfour weeks of rest, or five weeks of therapy followed by one, two,three, four or five weeks of rest, or administration once every twoweeks, once every three weeks or once a month. Treatment may be extendedfor any number of cycles, preferably at least 2, at least 4, at least 6,at least 8, at least 10, at least 12, at least 14, or at least 16cycles. The dosage may be up to 24 mg/kg. Exemplary dosages of use mayinclude 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg, 8mg/kg, 9 mg/kg, 10 mg/kg, 11 mg/kg, 12 mg/kg, 13 mg/kg, 14 mg/kg, 15mg/kg, 16 mg/kg, 17 mg/kg, and 18 mg/kg. Preferred dosages are 4, 6, 8,9, 10, or 12 mg/kg. The person of ordinary skill will realize that avariety of factors, such as age, general health, specific organ functionor weight, as well as effects of prior therapy on specific organ systems(e.g., bone marrow) may be considered in selecting an optimal dosage ofimmunoconjugate, and that the dosage and/or frequency of administrationmay be increased or decreased during the course of therapy. The dosagemay be repeated as needed, with evidence of tumor shrinkage observedafter as few as 4 to 8 doses. The optimized dosages and schedules ofadministration disclosed herein show unexpected superior efficacy andreduced toxicity in human subjects, which could not have been predictedfrom animal model studies. Surprisingly, the superior efficacy allowstreatment of tumors that were previously found to be resistant to one ormore standard anti-cancer therapies, including the parental compound,CPT-11 (irinotecan), from which SN-38 is derived in vivo.

The subject methods may include use of CT and/or PET/CT, or MM, tomeasure tumor response at regular intervals. Blood levels of tumormarkers, such as CEA (carcinoembryonic antigen), CA19-9, AFP, CA 15.3,or PSA, may also be monitored. Dosages and/or administration schedulesmay be adjusted as needed, according to the results of imaging and/ormarker blood levels.

A surprising result with the instant claimed compositions and methods isthe unexpected tolerability of high doses of antibody-drug conjugate,even with repeated infusions, with only relatively low-grade toxicitiesof nausea and vomiting observed, or manageable neutropenia. A furthersurprising result is the lack of accumulation of the antibody-drugconjugate, unlike other products that have conjugated SN-38 to albumin,PEG or other carriers. The lack of accumulation is associated withimproved tolerability and lack of serious toxicity even after repeatedor increased dosing. These surprising results allow optimization ofdosage and delivery schedule, with unexpectedly high efficacies and lowtoxicities. The claimed methods provide for shrinkage of solid tumors,in individuals with previously resistant cancers, of 15% or more,preferably 20% or more, preferably 30% or more, more preferably 40% ormore in size (as measured by longest diameter). The person of ordinaryskill will realize that tumor size may be measured by a variety ofdifferent techniques, such as total tumor volume, maximal tumor size inany dimension or a combination of size measurements in severaldimensions. This may be with standard radiological procedures, such ascomputed tomography, ultrasonography, and/or positron-emissiontomography. The means of measuring size is less important than observinga trend of decreasing tumor size with immunoconjugate treatment,preferably resulting in elimination of the tumor.

While the immunoconjugate may be administered as a periodic bolusinjection, in alternative embodiments the immunoconjugate may beadministered by continuous infusion of antibody-drug conjugates. Inorder to increase the Cmax and extend the PK of the immunoconjugate inthe blood, a continuous infusion may be administered for example byindwelling catheter. Such devices are known in the art, such asHICKMAN®, BROVIAC® or PORT-A-CATH® catheters (see, e.g., Skolnik et al.,Ther Drug Monit 32:741-48, 2010) and any such known indwelling cathetermay be used. A variety of continuous infusion pumps are also known inthe art and any such known infusion pump may be used. The dosage rangefor continuous infusion may be between 0.1 and 3.0 mg/kg per day. Morepreferably, these immunoconjugates can be administered by intravenousinfusions over relatively short periods of 2 to 5 hours, more preferably2-3 hours.

In particularly preferred embodiments, the immunoconjugates and dosingschedules may be efficacious in patients resistant to standardtherapies. For example, an anti-Trop-2 hRS7-SN-38 immunoconjugate may beadministered to a patient who has not responded to prior therapy withirinotecan, the parent agent of SN-38. Surprisingly, theirinotecan-resistant patient may show a partial or even a completeresponse to hRS7-SN-38. The ability of the immunoconjugate tospecifically target the tumor tissue may overcome tumor resistance byimproved targeting and enhanced delivery of the therapeutic agent. Otherantibody-SN-38 immunoconjugates may show similar improved efficacyand/or decreased toxicity, compared to alternative standard therapeutictreatments, and combinations of different SN-38 immunoconjugates, orSN-38-antibody conjugates in combination with an antibody conjugated toa radionuclide, toxin or other drug, may provide even more improvedefficacy and/or reduced toxicity. A specific preferred subject may be ametastatic colorectal cancer patient, metastatic pancreatic cancerpatient, a triple-negative breast cancer patient, a HER+, ER+,progesterone+breast cancer patient, a metastatic non-small-cell lungcancer (NSCLC) patient, a metastatic small-cell lung cancer patient, ametastatic stomach cancer patient, a metastatic renal cancer patient, ametastatic urinary bladder cancer patient, a metastatic ovarian cancerpatient, or a metastatic uterine cancer patient.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Preclinical in vivo therapy of athymic nude mice, bearing Capan1 human pancreatic carcinoma, with SN-38 conjugates of hRS7(anti-Trop-2), hPAM4 (anti-MUC5ac), hMN-14 (anti-CEACAM5) ornon-specific control hA20 (anti-CD20).

FIG. 2. Preclinical in vivo therapy of athymic nude mice, bearing BxPC3human pancreatic carcinoma, with anti-TROP2-CL2A-SN-38 conjugatescompared to controls.

FIG. 3. ADCC of various hRS7-ADCs vs. hRS7 IgG.

FIG. 4. (A) Structures of CL2-SN-38 and CL2A-SN-38. (B) Comparativeefficacy of anti-Trop-2 ADC linked to CL2 vs. CL2A linkers versus hA20ADC and saline control, using COLO 205 colonic adenocarcinoma. Animalswere treated twice weekly for 4 weeks as indicated by the arrows. COLO205 mice (N=6) were treated with 0.4 mg/kg ADC and tumors measured twicea week. (C) Comparative efficacy of anti-Trop-2 ADC linked to CL2 vs.CL2A linkers versus hA20 ADC and saline control, using Capan-1pancreatic adenocarcinoma. Animals were treated twice weekly for 4 weeksas indicated by the arrows. Capan-1 mice (N=10) were treated with 0.2mg/kg ADC and tumors measured weekly.

FIG. 5A. Therapeutic efficacy of hRS7-SN-38 ADC in several solidtumor-xenograft disease models. Efficacy of hRS7-CL2-SN-38 andhRS7-CL2A-SN-38 ADC treatment was studied in mice bearing humannon-small cell lung, colorectal, pancreatic, or squamous cell lung tumorxenografts. All the ADCs and controls were administered in the amountsindicated (expressed as amount of SN-38 per dose; long arrows=conjugateinjections, short arrows=irinotecan injections). Mice bearing Calu-3tumors (N=5-7) were injected with hRS7-CL2-SN-38 every 4 days for atotal of 4 injections (q4dx4).

FIG. 5B. Therapeutic efficacy of hRS7-SN-38 ADC in several solidtumor-xenograft disease models. Efficacy of hRS7-CL2-SN-38 andhRS7-CL2A-SN-38 ADC treatment was studied in mice bearing humannon-small cell lung, colorectal, pancreatic, or squamous cell lung tumorxenografts. All the ADCs and controls were administered in the amountsindicated (expressed as amount of SN-38 per dose; long arrows=conjugateinjections, short arrows=irinotecan injections). COLO 205 tumor-bearingmice (N=5) were injected 8 times (q4dx8) with the ADC or every 2 daysfor a total of 5 injections (q2dx5) with the MTD of irinotecan.

FIG. 5C. Therapeutic efficacy of hRS7-SN-38 ADC in several solidtumor-xenograft disease models. Efficacy of hRS7-CL2-SN-38 andhRS7-CL2A-SN-38 ADC treatment was studied in mice bearing humannon-small cell lung, colorectal, pancreatic, or squamous cell lung tumorxenografts. All the ADCs and controls were administered in the amountsindicated (expressed as amount of SN-38 per dose; long arrows=conjugateinjections, short arrows=irinotecan injections). Capan-1 (N=10) weretreated twice weekly for 4 weeks with the agents indicated.

FIG. 5D. Therapeutic efficacy of hRS7-SN-38 ADC in several solidtumor-xenograft disease models. Efficacy of hRS7-CL2-SN-38 andhRS7-CL2A-SN-38 ADC treatment was studied in mice bearing humannon-small cell lung, colorectal, pancreatic, or squamous cell lung tumorxenografts. All the ADCs and controls were administered in the amountsindicated (expressed as amount of SN-38 per dose; long arrows=conjugateinjections, short arrows=irinotecan injections). BxPC-3 tumor-bearingmice (N=10) were treated twice weekly for 4 weeks with the agentsindicated.

FIG. 5E. Therapeutic efficacy of hRS7-SN-38 ADC in several solidtumor-xenograft disease models. Efficacy of hRS7-CL2-SN-38 andhRS7-CL2A-SN-38 ADC treatment was studied in mice bearing humannon-small cell lung, colorectal, pancreatic, or squamous cell lung tumorxenografts. All the ADCs and controls were administered in the amountsindicated (expressed as amount of SN-38 per dose; long arrows=conjugateinjections, short arrows=irinotecan injections). In addition to ADCgiven twice weekly for 4 week, SK-MES-1 tumor-bearing (N=8) micereceived the MTD of CPT-11 (q2dx5).

FIG. 6A. Tolerability of hRS7-CL2A-SN-38 in Swiss-Webster mice.Fifty-six Swiss-Webster mice were administered 2 i.p. doses of buffer orthe hRS7-CL2A-SN-38 3 days apart (4, 8, or 12 mg/kg of SN-38 per dose;250, 500, or 750 mg conjugate protein/kg per dose). Seven and 15 daysafter the last injection, 7 mice from each group were euthanized, withblood counts and serum chemistries performed. Graphs show the percent ofanimals in each group that had elevated levels of AST.

FIG. 6B. Tolerability of hRS7-CL2A-SN-38 in Swiss-Webster mice.Fifty-six Swiss-Webster mice were administered 2 i.p. doses of buffer orthe hRS7-CL2A-SN-38 3 days apart (4, 8, or 12 mg/kg of SN-38 per dose;250, 500, or 750 mg conjugate protein/kg per dose). Seven and 15 daysafter the last injection, 7 mice from each group were euthanized, withblood counts and serum chemistries performed. Graphs show the percent ofanimals in each group that had elevated levels of ALT.

FIG. 6C. Tolerability of hRS7-CL2A-SN-38 in Cynomolgus monkeys. Sixmonkeys per group were injected twice 3 days apart with buffer (control)or hRS7-CL2A-SN-38 at 0.96 mg/kg or 1.92 mg/kg of SN-38 equivalents perdose (60 and 120 mg/kg conjugate protein). All animals were bled on day−1, 3, and 6. Four monkeys were bled on day 11 in the 0.96 mg/kg group,3 in the 1.92 mg/kg group. Changes in neutrophil counts in Cynomolgusmonkeys.

FIG. 6D. Tolerability of hRS7-CL2A-SN-38 in Cynomolgus monkeys. Sixmonkeys per group were injected twice 3 days apart with buffer (control)or hRS7-CL2A-SN-38 at 0.96 mg/kg or 1.92 mg/kg of SN-38 equivalents perdose (60 and 120 mg/kg conjugate protein). All animals were bled on day−1, 3, and 6. Four monkeys were bled on day 11 in the 0.96 mg/kg group,3 in the 1.92 mg/kg group. Changes in platelet counts in Cynomolgusmonkeys.

FIG. 7A. Comparison of in vitro efficacy of anti-Trop-2 ADCs (hRS7-SN-38versus MAB650-SN-38) in Capan-1 human pancreatic adenocarcinoma.

FIG. 7B. Comparison of in vitro efficacy of anti-Trop-2 ADCs (hRS7-SN-38versus MAB650-SN-38) in BxPC-3 human pancreatic adenocarcinoma.

FIG. 7C. Comparison of in vitro efficacy of anti-Trop-2 ADCs (hRS7-SN-38versus MAB650-SN-38) in NCI-N87 human gastric adenocarcinoma.

FIG. 8A. Comparison of in vitro efficacy of 162-46.2-SN-38 vs.hRS7-SN-38 in BxPC-3 human pancreatic adenocarcinoma cells.

FIG. 8B. Comparison of in vitro efficacy of 162-46.2-SN-38 vs.hRS7-SN-38 in MDA-MB-468 human breast adenocarcinoma cells.

FIG. 9. IMMU-132 phase I/II data for best response by RECIST criteria.

FIG. 10. IMMU-132 phase I/II data for time to progression and bestresponse (RECIST).

FIG. 11. Therapeutic efficacy of murine anti-Trop-2-SN-38 ADC(162-46.2-SN-38) compared to hRS7-SN-38 in mice bearing NCI-N87 humangastric carcinoma xenografts.

FIG. 12. Accumulation of SN-38 in tumors of nude mice with Capan-1 humanpancreatic cancer xenografts, when administered as free irinotecan vs.IMMU-132 ADC.

FIG. 13. Individual patient demographics and prior treatment for phaseI/II IMMU-132 anti-Trop-2 ADC in pancreatic cancer patients.

FIG. 14. Response assessment to IMMU-132 anti-Trop-2 ADC in pancreaticcancer patients.

FIG. 15. Summary of time to progression (TTP) results in humanpancreatic cancer patients administered IMMU-132 anti-Trop-2 ADC.

FIG. 16. Adverse events observed in phase I study of IMMU-132 in varioustumor types.

FIG. 17. Response assessment in sacituzumab govitecan-treated patients.(A) Composite schematic showing the best response (y-axis) determinedfrom target lesion measurements according to RECIST 1.1 and thetime-to-progression (z-axis; TTP expressed in months), measured from thedate of the first dose until CT documentation of progression as perRECIST. Best response bars are color-coded to identify the 4 startingdose levels. Four of the 25 patients (numbers 6, 9, 14, and 23; 2 PDC, 1GC and 1 SCLC) who were classified with disease progression are notshown because either they did not have a follow-up CT with measurementof target lesions or they had new lesions despite having stable targetlesions measurements. A bar break (//) shown for two PD patients denotestarget lesions increased >30%, whereas TTP values in the boxes at top ofthe graph show the patients who exceeded 9 months. The number of priortherapies (in parentheses) and the patients who received priortopoisomerase I therapy (asterisks) are indicated below the graph. (B)Graph showing the patients sorted according to survival, showing alsotheir TTP. Survival data were unavailable for 2 PDC patients (numbers 6and 17 with TTP 1.0 and 2.9 months).

FIG. 18. CT response assessment in 2 of 3 patients with >30% reductionin target lesions. Patient 22 is a 65-year-old woman with poorlydifferentiated SCLC (Trop-2 expression by immunohistology, 3+) who hadreceived 2 months of carboplatin/etoposide (topoisomerase-II inhibitor)and 1 month of topotecan (topoisomerase-I inhibitor) with no response,followed by local radiation for 6 weeks (3000 cGy), but progressed. Fourweeks later, the patient started sacituzumab govitecan at 12 mg/kg (2doses), which was reduced to 9.0 mg/kg (1 dose), and finally to 6.75mg/kg for 9 doses. The patient presented initially with the sum of thelongest diameters (SLD) of the target lesions totaling 19.3 cm. Two ofthe target lesions showing the best shrinkage are shown at baseline (Aand C). After 4 treatments, she had a 38% reduction in target lesions,including a substantial reduction in the main lung lesion (5.8 to 2.7cm; panels B and D). On her next CT assessment 12 weeks later, thepatient progressed. Patient 3 a 62 year-old woman, who 5 months afterher initial diagnosis and surgery for colon cancer had a hepaticresection for liver metastases and then received 7 months of treatmentwith FOLFOX and 1 month of only 5-fluorouracil. She was referred to thesacituzumab govitecan trial with multiple lesions, primarily in theliver (left panels A, C, and E). Immunohistology showed a 2+ staining ofher primary cancer; her plasma CEA was 781 ng/mL. Therapy was initiatedat 8 mg/kg and 6 treatments later (12 weeks), the 3 target lesions hadreduced from 7.9 cm to 5.0 cm (−37%; PR). The response was confirmed 6.6weeks later (after ten doses), with additional shrinkage to 3.8 cm(−52%). Panels B, D, and F show the shrinkage of these 3 lesions (59%reduction at this time) 32 weeks from the start of treatment and afterreceiving 18 doses. The patient continued therapy, achieving a maximumtumor reduction of 65% 10 months after treatment was initiated (28doses). Plasma CEA decreased to 26.5 ng/mL after 18 doses, butthereafter began to increase despite continued radiological evidence(target and non-target lesions) of additional disease reduction orstabilization. Approximately 1 year from the start of treatment (31doses given), one of the 3 target lesions progressed.

FIG. 19. Therapeutic efficacy of IMMU-132 with different DARs. NCI-N87human gastric carcinoma xenografts (subcutaneous) were established asdescribed in Methods. (A) Four groups of mice (N=9) were injected IVwith 2×0.5 mg (arrows) of IMMU-132 conjugates prepared with a DAR=6.89,3.28, or 1.64. Control animals received saline. Therapy began 7 daysafter tumor cells were administered (size was 0.248±0.047 cm³). Survivalcurves were generated based on the time to progression to >1.0 cm³, andwere analyzed by log-rank test (significance at P<0.05). (B) NCI-N87tumor-bearing mice (N=7-9; starting size=0.271±0.053 cm³) were treatedwith either 0.5 mg IMMU-132 (DAR=6.89) or 1.0 mg DAR=3.28 twice weeklyfor two weeks (arrows). Mice were euthanized and deemed to havesuccumbed to disease once tumors grew to >1.0 cm³. Profiles ofindividual tumor growth were obtained through linear-curve modeling.Statistical analysis of tumor growth was based on area under the curve(AUC) performed up to the time that the first animal within a group waseuthanized due to disease progression. An f-test was employed todetermine equality of variance between groups prior to statisticalanalysis of growth curves. A two-tailed t-test was used to assessstatistical significance between the various treatment groups andcontrols, except for the saline control, where a one-tailed t-test wasused (significance at P≤0.05).

FIG. 20. Therapeutic efficacy of IMMU-132 in TNBC xenograft models. (A)Twenty-two days after s.c. implantation of MDA-MB-468 tumors (at theonset of treatment, tumors averaged 0.223±0.055 cm³), nude mice (7-8 pergroup) were injected IV with IMMU-132 or a control hA20 anti-CD20-SN-38conjugate twice weekly for two weeks (0.12 or 0.20 mg/kg SN-38equivalents per dose). Other animals were given irinotecan (10mg/kg/dose; SN-38 equivalent based on mass=5.8 mg/kg) IV every other dayfor 10 days for a total of five injections. (B) Starting on day 56 aftertreatment initiation, all animals in the control hA20-SN-38 group weregiven IMMU-132 (4×0.2 mg/kg SN-38 equivalents). The size of the tumorsin the individual animals of this group from the onset of tumortransplantation is given. Red arrows indicate when the hA20-SN-38conjugate was first given, and purple arrows indicate when the treatmentwith IMMU-132 was initiated. (C) Mice (N=12) bearing the MDA-MB-231 TNBCcell line (0.335±0.078 cm³) were treated with IMMU-132 or the controlhA20-SN-38 conjugate (0.4 mg/kg SN-38 equivalents), irinotecan (6.5mg/kg; ˜3.8 mg/kg SN-38 equivalents), or a combination of hRS7 IgG (25mg/kg) plus irinotecan (6.5 mg/kg).

FIG. 21. ADCC activity of IMMU-132. Specific cell lysis of target cellsby human PBMCs mediated by IMMU-132 was compared to parental hRS7.Target cells were plated the night before and the assay performed asdescribed in the Examples below. (A) MDA-MB-468 target cells. (B)NIH:OVCAR-3 target cells. (C) BxPC-3 target cells. *hRS7 versus all theother test agents (P<0.0054). **IMMU-132 versus negative controlshLL2-SN-38 and hLL2 (P<0.0003). *** IMMU-132 versus negative controlhLL2-CL2ASN-38 (P<0.0019).

FIG. 22. Pharmacokinetics of IMMU-132 in mice. Naive nude mice (N=5)were injected i.v. with 200 μg of IMMU-132. At various time-points thesemice were bled and serum obtained and analyzed for intact conjugate andcarrier hRS7 antibody, as described in the Examples below. Forcomparison, another group of mice was injected with 200 μg parentalhRS7. (A) Serum concentration and clearance of hRS7 from parentalcontrol injected mice. Concentration and clearance of (B) hRS7 carrierantibody versus (C) intact conjugate from IMMU-132 injected mice.Graphed data shown as mean±S.D.

FIG. 23. Efficacy of IMMU-132 in mice bearing human gastric carcinomaxenograft. Mice bearing NCI-N87 human gastric tumors (TV=0.249±0.049cm³) were treated with 0.35 mg IMMU-132 twice weekly for four weeks. (A)Mean tumor growth curves for IMMU-132 treated animals compared to salineand non-tumor-targeting control ADC, hA20-CL2A-SN-38, treated mice.Arrows indicate therapy days. (B) Survival curves for treated mice witha disease end-point of tumor progression greater than 1.0 cm³.

FIG. 24. Various IMMU-132 dosing schemes in mice bearing pancreatic andgastric tumor xenografts. Nude mice (N=8-10) bearing s.c. BxPC-3 orNCI-N87 xenografts were prepared. (A) BxPC-3-bearing mice were treated(arrows) with two cycles of IMMU-132 at 1 mg every 14 d, 0.5 mg weeklyfor two weeks, or 0.25 mg twice weekly for two weeks, totaling 2 mgIMMU-132 to all the mice. (B) Similar dosing of NCIN87-bearing mice(arrows), with mice in the 1-mg treatment group receiving one additionalcycle. (C) Chronic dosing of IMMU-132 in mice bearing NCI-N87, using0.5-mg once weekly for 2 weeks in a 3-week treatment cycle for a totalof 4 cycles. Corresponding survival curves (end-point: tumorprogression >1.0 cm3) to right of each tumor growth curve.

FIG. 25. Responses in 52 human TNBC patients treated with 10 mg/kgIMMU-132, after failing numerous prior therapies.

FIG. 26. Time to progression for CR+PR+SD in TNBC patients treated with10 mg/kg IMMU-132.

FIG. 27. Progression-free survival in TNBC patients treated with 10mg/kg IMMU-132.

FIG. 28. Best response in 29 assessable human NSCLC patients treatedwith 8 to 10 mg/kg IMMU-132.

FIG. 29. Time to progression in NSCLC patients treated with 8-10 mg/kgIMMU-132.

FIG. 30. Progression-free survival in NSCLC patients treated with 8 or10 mg/kg IMMU-132.

FIG. 31. Best response in 25 assessable human SCLC patients treated with8 to 10 mg/kg IMMU-132.

FIG. 32. Time to progression in SCLC patients treated with 8-10 mg/kgIMMU-132.

FIG. 33. Progression-free survival in SCLC patients treated with 8 or 10mg/kg IMMU-132.

FIG. 34. Best response in 11 assessable human urothelial cancer patientstreated with IMMU-132.

FIG. 35. Time to progression in urothelial cancer patients treated withIMMU-132.

DETAILED DESCRIPTION OF THE INVENTION Definitions

In the description that follows, a number of terms are used and thefollowing definitions are provided to facilitate understanding of theclaimed subject matter. Terms that are not expressly defined herein areused in accordance with their plain and ordinary meanings.

Unless otherwise specified, a or an means “one or more.”

The term about is used herein to mean plus or minus ten percent (10%) ofa value. For example, “about 100” refers to any number between 90 and110.

An antibody, as used herein, refers to a full-length (i.e., naturallyoccurring or formed by normal immunoglobulin gene fragmentrecombinatorial processes) immunoglobulin molecule (e.g., an IgGantibody) or an antigen-binding portion of an immunoglobulin molecule,such as an antibody fragment. An antibody or antibody fragment may beconjugated or otherwise derivatized within the scope of the claimedsubject matter. Such antibodies include but are not limited to IgG1,IgG2, IgG3, IgG4 (and IgG4 subforms), as well as IgA isotypes. As usedbelow, the abbreviation “MAb” may be used interchangeably to refer to anantibody, antibody fragment, monoclonal antibody or multispecificantibody.

An antibody fragment is a portion of an antibody such as F(ab′)2,F(ab)₂, Fab′, Fab, Fv, scFv (single chain Fv), single domain antibodies(DABs or VHHs) and the like, including the half-molecules of IgG4 citedabove (van der Neut Kolfschoten et al. (Science 2007; 317(14September):1554-1557). Regardless of structure, an antibody fragment ofuse binds with the same antigen that is recognized by the intactantibody. The term “antibody fragment” also includes synthetic orgenetically engineered proteins that act like an antibody by binding toa specific antigen to form a complex. For example, antibody fragmentsinclude isolated fragments consisting of the variable regions, such asthe “Fv” fragments consisting of the variable regions of the heavy andlight chains and recombinant single chain polypeptide molecules in whichlight and heavy variable regions are connected by a peptide linker(“scFv proteins”). The fragments may be constructed in different ways toyield multivalent and/or multispecific binding forms.

A naked antibody is generally an entire antibody that is not conjugatedto a therapeutic agent. A naked antibody may exhibit therapeutic and/orcytotoxic effects, for example by Fc-dependent functions, such ascomplement fixation (CDC) and ADCC (antibody-dependent cellcytotoxicity). However, other mechanisms, such as apoptosis,anti-angiogenesis, anti-metastatic activity, anti-adhesion activity,inhibition of heterotypic or homotypic adhesion, and interference insignaling pathways, may also provide a therapeutic effect. Nakedantibodies include polyclonal and monoclonal antibodies, naturallyoccurring or recombinant antibodies, such as chimeric, humanized orhuman antibodies and fragments thereof. In some cases a “naked antibody”may also refer to a “naked” antibody fragment. As defined herein,“naked” is synonymous with “unconjugated,” and means not linked orconjugated to a therapeutic agent.

A chimeric antibody is a recombinant protein that contains the variabledomains of both the heavy and light antibody chains, including thecomplementarity determining regions (CDRs) of an antibody derived fromone species, preferably a rodent antibody, more preferably a murineantibody, while the constant domains of the antibody molecule arederived from those of a human antibody. For veterinary applications, theconstant domains of the chimeric antibody may be derived from that ofother species, such as a primate, cat or dog.

A humanized antibody is a recombinant protein in which the CDRs from anantibody from one species; e.g., a murine antibody, are transferred fromthe heavy and light variable chains of the murine antibody into humanheavy and light variable domains (framework regions). The constantdomains of the antibody molecule are derived from those of a humanantibody. In some cases, specific residues of the framework region ofthe humanized antibody, particularly those that are touching or close tothe CDR sequences, may be modified, for example replaced with thecorresponding residues from the original murine, rodent, subhumanprimate, or other antibody.

A human antibody is an antibody obtained, for example, from transgenicmice that have been “engineered” to produce human antibodies in responseto antigenic challenge. In this technique, elements of the human heavyand light chain loci are introduced into strains of mice derived fromembryonic stem cell lines that contain targeted disruptions of theendogenous heavy chain and light chain loci. The transgenic mice cansynthesize human antibodies specific for various antigens, and the micecan be used to produce human antibody-secreting hybridomas. Methods forobtaining human antibodies from transgenic mice are described by Greenet al., Nature Genet. 7:13 (1994), Lonberg et al., Nature 368:856(1994), and Taylor et al., Int. Immun. 6:579 (1994). A fully humanantibody also can be constructed by genetic or chromosomal transfectionmethods, as well as phage display technology, all of which are known inthe art. See for example, McCafferty et al., Nature 348:552-553 (1990)for the production of human antibodies and fragments thereof in vitro,from immunoglobulin variable domain gene repertoires from unimmunizeddonors. In this technique, human antibody variable domain genes arecloned in-frame into either a major or minor coat protein gene of afilamentous bacteriophage, and displayed as functional antibodyfragments on the surface of the phage particle. Because the filamentousparticle contains a single-stranded DNA copy of the phage genome,selections based on the functional properties of the antibody alsoresult in selection of the gene encoding the antibody exhibiting thoseproperties. In this way, the phage mimics some of the properties of theB cell. Phage display can be performed in a variety of formats, fortheir review, see e.g. Johnson and Chiswell, Current Opinion inStructural Biology 3:5564-571 (1993). Human antibodies may also begenerated by in vitro activated B cells. See U.S. Pat. Nos. 5,567,610and 5,229,275, the Examples section of each of which is incorporatedherein by reference.

A therapeutic agent is an atom, molecule, or compound that is useful inthe treatment of a disease. Examples of therapeutic agents include, butare not limited to, antibodies, antibody fragments, immunoconjugates,drugs, cytotoxic agents, pro-apopoptotic agents, toxins, nucleases(including DNAses and RNAses), hormones, immunomodulators, chelators,boron compounds, photoactive agents or dyes, radionuclides,oligonucleotides, interference RNA, siRNA, RNAi, anti-angiogenic agents,chemotherapeutic agents, cyokines, chemokines, prodrugs, enzymes,binding proteins or peptides or combinations thereof.

An immunoconjugate is an antibody, antigen-binding antibody fragment,antibody complex or antibody fusion protein that is conjugated to atherapeutic agent. Conjugation may be covalent or non-covalent.Preferably, conjugation is covalent.

As used herein, the term antibody fusion protein is arecombinantly-produced antigen-binding molecule in which one or morenatural antibodies, single-chain antibodies or antibody fragments arelinked to another moiety, such as a protein or peptide, a toxin, acytokine, a hormone, etc. In certain preferred embodiments, the fusionprotein may comprise two or more of the same or different antibodies,antibody fragments or single-chain antibodies fused together, which maybind to the same epitope, different epitopes on the same antigen, ordifferent antigens.

An immunomodulator is a therapeutic agent that when present, alters,suppresses or stimulates the body's immune system. Typically, animmunomodulator of use stimulates immune cells to proliferate or becomeactivated in an immune response cascade, such as macrophages, dendriticcells, B-cells, and/or T-cells. However, in some cases animmunomodulator may suppress proliferation or activation of immunecells. An example of an immunomodulator as described herein is acytokine, which is a soluble small protein of approximately 5-20 kDathat is released by one cell population (e.g., primed T-lymphocytes) oncontact with specific antigens, and which acts as an intercellularmediator between cells. As the skilled artisan will understand, examplesof cytokines include lymphokines, monokines, interleukins, and severalrelated signaling molecules, such as tumor necrosis factor (TNF) andinterferons. Chemokines are a subset of cytokines. Certain interleukinsand interferons are examples of cytokines that stimulate T cell or otherimmune cell proliferation. Exemplary interferons include interferon-α,interferon-β, interferon-γ and interferon-λ.

CPT is an abbreviation for camptothecin, and as used in the presentapplication CPT represents camptothecin itself or an analog orderivative of camptothecin, such as SN-38. The structures ofcamptothecin and some of its analogs, with the numbering indicated andthe rings labeled with letters A-E, are given in formula 1 in Chart 1below.

Anti-Trop-2 Antibodies

Preferably, the subject ADCs include at least one antibody or fragmentthereof that binds to Trop-2. In a specific preferred embodiment, theanti-Trop-2 antibody may be a humanized RS7 antibody (see, e.g., U.S.Pat. No. 7,238,785, incorporated herein by reference in its entirety),comprising the light chain CDR sequences CDR1 (KASQDVSIAVA, SEQ IDNO:21); CDR2 (SASYRYT, SEQ ID NO:22); and CDR3 (QQHYITPLT, SEQ ID NO:23)and the heavy chain CDR sequences CDR1 (NYGMN, SEQ ID NO:24); CDR2(WINTYTGEPTYTDDFKG, SEQ ID NO:25) and CDR3 (GGFGSSYWYFDV, SEQ ID NO:26).

The RS7 antibody was a murine IgG₁ raised against a crude membranepreparation of a human primary squamous cell lung carcinoma. (Stein etal., Cancer Res. 50: 1330, 1990) The RS7 antibody recognizes a 46-48 kDaglycoprotein, characterized as cluster 13. (Stein et al., Int. J. CancerSupp. 8:98-102, 1994) The antigen was designated as EGP-1 (epithelialglycoprotein-1), but is also referred to as Trop-2.

Trop-2 is a type-I transmembrane protein and has been cloned from bothhuman (Fornaro et al., Int J Cancer 1995; 62:610-8) and mouse cells(Sewedy et al., Int J Cancer 1998; 75:324-30). In addition to its roleas a tumor-associated calcium signal transducer (Ripani et al., Int JCancer 1998; 76:671-6), the expression of human Trop-2 was shown to benecessary for tumorigenesis and invasiveness of colon cancer cells,which could be effectively reduced with a polyclonal antibody againstthe extracellular domain of Trop-2 (Wang et al., Mol Cancer Ther 2008;7:280-5).

The growing interest in Trop-2 as a therapeutic target for solid cancers(Cubas et al., Biochim Biophys Acta 2009; 1796:309-14) is attested byfurther reports that documented the clinical significance ofoverexpressed Trop-2 in breast (Huang et al., Clin Cancer Res 2005;11:4357-64), colorectal (Ohmachi et al., Clin Cancer Res 2006;12:3057-63; Fang et al., Int J Colorectal Dis 2009; 24:875-84), and oralsquamous cell (Fong et al., Modern Pathol 2008; 21:186-91) carcinomas.The latest evidence that prostate basal cells expressing high levels ofTrop-2 are enriched for in vitro and in vivo stem-like activity isparticularly noteworthy (Goldstein et al., Proc Natl Acad Sci USA 2008;105:20882-7).

Flow cytometry and immunohistochemical staining studies have shown thatthe RS7 MAb detects antigen on a variety of tumor types, with limitedbinding to normal human tissue (Stein et al., 1990). Trop-2 is expressedprimarily by carcinomas such as carcinomas of the lung, stomach, urinarybladder, breast, ovary, uterus, and prostate. Localization and therapystudies using radiolabeled murine RS7 MAb in animal models havedemonstrated tumor targeting and therapeutic efficacy (Stein et al.,1990; Stein et al., 1991).

Strong RS7 staining has been demonstrated in tumors from the lung,breast, bladder, ovary, uterus, stomach, and prostate. (Stein et al.,Int. J. Cancer 55:938, 1993) The lung cancer cases comprised bothsquamous cell carcinomas and adenocarcinomas. (Stein et al., Int. J.Cancer 55:938, 1993) Both cell types stained strongly, indicating thatthe RS7 antibody does not distinguish between histologic classes ofnon-small-cell carcinoma of the lung.

The RS7 MAb is rapidly internalized into target cells (Stein et al.,1993). The internalization rate constant for RS7 MAb is intermediatebetween the internalization rate constants of two other rapidlyinternalizing MAbs, which have been demonstrated to be useful forimmunoconjugate production. (Id.) It is well documented thatinternalization of immunoconjugates is a requirement for anti-tumoractivity. (Pastan et al., Cell 47:641, 1986) Internalization of drugimmunoconjugates has been described as a major factor in anti-tumorefficacy. (Yang et al., Proc. Nat'l Acad. Sci. USA 85: 1189, 1988) Thus,the RS7 antibody exhibits several important properties for therapeuticapplications.

While the hRS7 antibody is preferred, other anti-Trop-2 antibodies areknown and/or publicly available and in alternative embodiments may beutilized in the subject ADCs. While humanized or human antibodies arepreferred for reduced immunogenicity, in alternative embodiments achimeric antibody may be of use. As discussed below, methods of antibodyhumanization are well known in the art and may be utilized to convert anavailable murine or chimeric antibody into a humanized form.

Anti-Trop-2 antibodies are commercially available from a number ofsources and include LS-C126418, LS-C178765, LS-C126416, LS-C126417(LifeSpan BioSciences, Inc., Seattle, Wash.); 10428-MM01, 10428-MM02,10428-R001, 10428-R030 (Sino Biological Inc., Beijing, China); MR54(eBioscience, San Diego, Calif.); sc-376181, sc-376746, Santa CruzBiotechnology (Santa Cruz, Calif.); MM0588-49D6, (Novus Biologicals,Littleton, Colo.); ab79976, and ab89928 (ABCAM®, Cambridge, Mass.).

Other anti-Trop-2 antibodies have been disclosed in the patentliterature. For example, U.S. Publ. No. 2013/0089872 disclosesanti-Trop-2 antibodies K5-70 (Accession No. FERM BP-11251), K5-107(Accession No. FERM BP-11252), K5-116-2-1 (Accession No. FERM BP-11253),T6-16 (Accession No. FERM BP-11346), and T5-86 (Accession No. FERMBP-11254), deposited with the International Patent Organism Depositary,Tsukuba, Japan. U.S. Pat. No. 5,840,854 disclosed the anti-Trop-2monoclonal antibody BR110 (ATCC No. HB11698). U.S. Pat. No. 7,420,040disclosed an anti-Trop-2 antibody produced by hybridoma cell lineAR47A6.4.2, deposited with the IDAC (International Depository Authorityof Canada, Winnipeg, Canada) as accession number 141205-05. U.S. Pat.No. 7,420,041 disclosed an anti-Trop-2 antibody produced by hybridomacell line AR52A301.5, deposited with the IDAC as accession number141205-03. U.S. Publ. No. 2013/0122020 disclosed anti-Trop-2 antibodies3E9, 6G11, 7E6, 15E2, 18B1. Hybridomas encoding a representativeantibody were deposited with the American Type Culture Collection(ATCC), Accession Nos. PTA-12871 and PTA-12872. Immunoconjugate PF06263507, comprising an anti-5T4 (anti-Trop-2) antibody attached to thetubulin inhibitor monomethylauristatin F (MMAF) is available fromPfizer, Inc. (Groton, Conn.) (see, e.g., Sapra et al., 2013, Mol CancerTher 12:38-47). U.S. Pat. No. 8,715,662 discloses anti-Trop-2 antibodiesproduced by hybridomas deposited at the AID-ICLC (Genoa, Italy) withdeposit numbers PD 08019, PD 08020 and PD 08021. U.S. Patent ApplicationPubl. No. 20120237518 discloses anti-Trop-2 antibodies 77220, KM4097 andKM4590. U.S. Pat. No. 8,309,094 (Wyeth) discloses antibodies A1 and A3,identified by sequence listing. The Examples section of each patent orpatent application cited above in this paragraph is incorporated hereinby reference. Non-patent publication Lipinski et al. (1981, Proc Natl.Acad Sci USA, 78:5147-50) disclosed anti-Trop-2 antibodies 162-25.3 and162-46.2.

Numerous anti-Trop-2 antibodies are known in the art and/or publiclyavailable. As discussed below, methods for preparing antibodies againstknown antigens were routine in the art. The sequence of the human Trop-2protein was also known in the art (see, e.g., GenBank Accession No.CAA54801.1). Methods for producing humanized, human or chimericantibodies were also known. The person of ordinary skill, reading theinstant disclosure in light of general knowledge in the art, would havebeen able to make and use the genus of anti-Trop-2 antibodies in thesubject ADCs.

Use of antibodies against targets related to Trop-2 has been disclosedfor immunotherapeutics other than ADCs. The murine anti-Trop-1 IgG2aantibody edrecolomab (PANOREX®) has been used for treatment ofcolorectal cancer, although the murine antibody is not well suited forhuman clinical use (Baeuerle & Gires, 2007, Br. J Cancer 96:417-423).Low-dose subcutaneous administration of ecrecolomab was reported toinduce humoral immune responses against the vaccine antigen (Baeuerle &Gires, 2007). Adecatumumab (MT201), a fully human anti-Trop-1 antibody,has been used in metastatic breast cancer and early-stage prostatecancer and is reported to act through ADCC and CDC activity (Baeuerle &Gires, 2007). MT110, a single-chain anti-Trop-1/anti-CD3 bispecificantibody construct has reported efficacy against ovarian cancer(Baeuerle & Gires, 2007). Proxinium, an immunotoxin comprisinganti-Trop-1 single-chain antibody fused to Pseudomonas exotoxin, hasbeen tested in head-and-neck and bladder cancer (Baeuerle & Gires,2007). None of these studies contained any disclosure of the use ofanti-Trop-2 immunoconjugates or of drug-conjugated antibodies.

Camptothecin Conjugates

Non-limiting methods and compositions for preparing immunoconjugatescomprising a camptothecin therapeutic agent attached to an antibody orantigen-binding antibody fragment are described below. In preferredembodiments, the solubility of the drug is enhanced by placing a definedpolyethyleneglycol (PEG) moiety (i.e., a PEG containing a defined numberof monomeric units) between the drug and the antibody, wherein thedefined PEG is a low molecular weight PEG, preferably containing 1-30monomeric units, more preferably containing 1-12 monomeric units.

Preferably, a first linker connects the drug at one end and mayterminate with an acetylene or an azide group at the other end. Thisfirst linker may comprise a defined PEG moiety with an azide oracetylene group at one end and a different reactive group, such ascarboxylic acid or hydroxyl group, at the other end. Said bifunctionaldefined PEG may be attached to the amine group of an amino alcohol, andthe hydroxyl group of the latter may be attached to the hydroxyl groupon the drug in the form of a carbonate. Alternatively, the non-azide (oracetylene) moiety of said defined bifunctional PEG is optionallyattached to the N-terminus of an L-amino acid or a polypeptide, with theC-terminus attached to the amino group of amino alcohol, and the hydroxygroup of the latter is attached to the hydroxyl group of the drug in theform of carbonate or carbamate, respectively.

A second linker, comprising an antibody-coupling group and a reactivegroup complementary to the azide (or acetylene) group of the firstlinker, namely acetylene (or azide), may react with the drug-(firstlinker) conjugate via acetylene-azide cycloaddition reaction to furnisha final bifunctional drug product that is useful for conjugating todisease-targeting antibodies. The antibody-coupling group is preferablyeither a thiol or a thiol-reactive group.

Methods for selective regeneration of the 10-hydroxyl group in thepresence of the C-20 carbonate in preparations of drug-linker precursorinvolving CPT analogs such as SN-38 are provided below. Other protectinggroups for reactive hydroxyl groups in drugs such as the phenolichydroxyl in SN-38, for example t-butyldimethylsilyl ort-butyldiphenylsilyl, may also be used, and these are deprotected bytetrabutylammonium fluoride prior to linking of the derivatized drug toan antibody-coupling moiety. The 10-hydroxyl group of CPT analogs isalternatively protected as an ester or carbonate, other than ‘BOC’, suchthat the bifunctional CPT is conjugated to an antibody without priordeprotection of this protecting group. The protecting group is readilydeprotected under physiological pH conditions after the bioconjugate isadministered.

In the acetylene-azide coupling, referred to as ‘click chemistry’, theazide part may be on L2 with the acetylene part on L3. Alternatively, L2may contain acetylene, with L3 containing azide. ‘Click chemistry’refers to a copper (+1)-catalyzed cycloaddition reaction between anacetylene moiety and an azide moiety (Kolb H C and Sharpless K B, DrugDiscov Today 2003; 8: 1128-37), although alternative forms of clickchemistry are known and may be used. Click chemistry takes place inaqueous solution at near-neutral pH conditions, and is thus amenable fordrug conjugation. The advantage of click chemistry is that it ischemoselective, and complements other well-known conjugation chemistriessuch as the thiol-maleimide reaction.

While the present application focuses on use of antibodies or antibodyfragments as targeting moieties, the skilled artisan will realize thatwhere a conjugate comprises an antibody or antibody fragment, anothertype of targeting moiety, such as an aptamer, avimer, affibody orpeptide ligand, may be substituted.

An exemplary preferred embodiment is directed to a conjugate of a drugderivative and an antibody of the general formula 2,

MAb-[L2]-[L1]-[AA]_(m)-[A′]-Drug  (2)

where MAb is a disease-targeting antibody; L2 is a component of thecross-linker comprising an antibody-coupling moiety and one or more ofacetylene (or azide) groups; L1 comprises a defined PEG with azide (oracetylene) at one end, complementary to the acetylene (or azide) moietyin L2, and a reactive group such as carboxylic acid or hydroxyl group atthe other end; AA is an L-amino acid; m is an integer with values of 0,1, 2, 3, or 4; and A′ is an additional spacer, selected from the groupof ethanolamine, 4-hydroxybenzyl alcohol, 4-aminobenzyl alcohol, orsubstituted or unsubstituted ethylenediamine. The L amino acids of ‘AA’are selected from alanine, arginine, asparagine, aspartic acid,cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine,leucine, lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine, and valine. If the A′ group contains hydroxyl, itis linked to the hydroxyl group or amino group of the drug in the formof a carbonate or carbamate, respectively.

In a preferred embodiment of formula 2, A′ is a substituted ethanolaminederived from an L-amino acid, wherein the carboxylic acid group of theamino acid is replaced by a hydroxymethyl moiety. A′ may be derived fromany one of the following L-amino acids: alanine, arginine, asparagine,aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine,isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine,threonine, tryptophan, tyrosine, and valine.

In an example of the conjugate of the preferred embodiment of formula 2,m is 0, A′ is L-valinol, and the drug is exemplified by SN-38. Theresultant structure is shown in formula 3.

In another example of the conjugate of the preferred embodiment offormula 2, m is 1 and represented by a derivatized L-lysine, A′ isL-valinol, and the drug is exemplified by SN-38. The structure is shownin formula 4.

In this embodiment, an amide bond is first formed between the carboxylicacid of an amino acid such as lysine and the amino group of valinol,using orthogonal protecting groups for the lysine amino groups. Theprotecting group on the N-terminus of lysine is removed, keeping theprotecting group on the side chain of lysine intact, and the N-terminusis coupled to the carboxyl group on the defined PEG with azide (oracetylene) at the other end. The hydroxyl group of valinol is thenattached to the 20-chloroformate derivative of 10-hydroxy-protectedSN-38, and this intermediate is coupled to an L2 component carrying theantibody-binding moiety as well as the complementary acetylene (orazide) group involved in the click cycloaddition chemistry. Finally,removal of protecting groups at both lysine side chain and SN-38 givesthe product of this example, shown in formula 3.

While not wishing to be bound by theory, the small MW SN-38 product,namely valinol-SN-38 carbonate, generated after intracellularproteolysis, has the additional pathway of liberation of intact SN-38through intramolecular cyclization involving the amino group of valinoland the carbonyl of the carbonate.

In another preferred embodiment, A′ of the general formula 2 is A-OH,whereby A-OH is a collapsible moiety such as 4-aminobenzyl alcohol or asubstituted 4-aminobenzyl alcohol substituted with a C₁-C₁₀ alkyl groupat the benzylic position, and the latter, via its amino group, isattached to an L-amino acid or a polypeptide comprising up to fourL-amino acid moieties; wherein the N-terminus is attached to across-linker terminating in the antibody-binding group.

An example of a preferred embodiment is given below, wherein the A-OHembodiment of A′ of general formula (2) is derived from substituted4-aminobenzyl alcohol, and ‘AA’ is comprised of a single L-amino acidwith m=1 in the general formula (2), and the drug is exemplified withSN-38. The structure is represented below (formula 5, referred to asMAb-CLX-SN-38). Single amino acid of AA is selected from any one of thefollowing L-amino acids: alanine, arginine, asparagine, aspartic acid,cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine,leucine, lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine, and valine. The substituent R on 4-aminobenzylalcohol moiety (A-OH embodiment of A′) is hydrogen or an alkyl groupselected from C1-C10 alkyl groups.

A particularly preferred embodiment of MAb-CLX-SN-38 of formula 5,wherein the single amino acid AA is L-lysine and R═H, and the drug isexemplified by SN-38 (formula 6; referred to as MAb-CL2A-SN-38). Thestructure differs from the linker MAb-CL2-SN-38 in the substitution of asingle lysine residue for a Phe-Lys dipeptide found in the CL2 linker.The Phe-Lys dipeptide was designed as a cathepsin B cleavage site forlysosomal enzyme, which was considered to be important for intracellularrelease of bound drug. Surprisingly, despite the elimination of thecathepsin-cleavage site, immunoconjugates comprising a CL2A linker areat least as efficacious, and may be more efficacious in vivo than thosecomprising a CL2 linker.

Other embodiments are possible within the context of10-hydroxy-containing camptothecins, such as SN-38. In the example ofSN-38 as the drug, the more reactive 10-hydroxy group of the drug isderivatized leaving the 20-hydroxyl group unaffected. Within the generalformula 2, A′ is a substituted ethylenediamine. An example of thisembodiment is represented by the formula ‘7’ below, wherein the phenolichydroxyl group of SN-38 is derivatized as a carbamate with a substitutedethylenediamine, with the other amine of the diamine derivatized as acarbamate with a 4-aminobenzyl alcohol, and the latter's amino group isattached to Phe-Lys dipeptide. In this structure (formula 7), R and R′are independently hydrogen or methyl. It is referred to asMAb-CL17-SN-38 or MAb-CL2E-SN-38, when R=R′=methyl.

In certain embodiments, AA comprises a polypeptide moiety, preferably adi, tri or tetrapeptide, that is cleavable by intracellular peptidase.Examples are: Ala-Leu, Leu-Ala-Leu, and Ala-Leu-Ala-Leu (SEQ ID NO: 57)(Trouet et al., 1982). Another example is a Phe-Lys moiety that iscleavable by lysosomal cathepsin.

In a preferred embodiment, the L1 component of the conjugate contains adefined polyethyleneglycol (PEG) spacer with 1-30 repeating monomericunits. In a further preferred embodiment, PEG is a defined PEG with 1-12repeating monomeric units. The introduction of PEG may involve usingheterobifunctionalized PEG derivatives which are available commercially.The heterobifunctional PEG may contain an azide or acetylene group. Anexample of a heterobifunctional defined PEG containing 8 repeatingmonomeric units, with ‘NHS’ being succinimidyl, is given below informula 8:

In a preferred embodiment, L2 has a plurality of acetylene (or azide)groups, ranging from 2-40, but preferably 2-20, and more preferably 2-5,and a single antibody-binding moiety.

A representative SN-38 conjugate of an antibody containing multiple drugmolecules and a single antibody-binding moiety is shown below. The ‘L2’component of this structure is appended to 2 acetylenic groups,resulting in the attachment of two azide-appended SN-38 molecules. Thebonding to MAb is represented as a succinimide.

In preferred embodiments, when the bifunctional drug contains athiol-reactive moiety as the antibody-binding group, the thiols on theantibody are generated on the lysine groups of the antibody using athiolating reagent. Methods for introducing thiol groups onto antibodiesby modifications of MAb's lysine groups are well known in the art (Wongin Chemistry of protein conjugation and cross-linking, CRC Press, Inc.,Boca Raton, Fla. (1991), pp 20-22). Alternatively, mild reduction ofinterchain disulfide bonds on the antibody (Willner et al., BioconjugateChem. 4:521-527 (1993)) using reducing agents such as dithiothreitol(DTT) can generate 7-to-10 thiols on the antibody; which has theadvantage of incorporating multiple drug moieties in the interchainregion of the MAb away from the antigen-binding region. In a morepreferred embodiment, attachment of SN-38 to reduced disulfidesulfhydryl groups results in formation of an antibody-SN-38immunoconjugate with 6 SN-38 moieties covalently attached per antibodymolecule. Other methods of providing cysteine residues for attachment ofdrugs or other therapeutic agents are known, such as the use of cysteineengineered antibodies (see U.S. Pat. No. 7,521,541, the Examples sectionof which is incorporated herein by reference.)

In alternative preferred embodiments, the chemotherapeutic moiety isselected from the group consisting of doxorubicin (DOX), epirubicin,morpholinodoxorubicin (morpholino-DOX), cyanomorpholino-doxorubicin(cyanomorpholino-DOX), 2-pyrrolino-doxorubicin (2-PDOX), Pro-2PDOX, CPT,10-hydroxy camptothecin, SN-38, topotecan, lurtotecan,9-aminocamptothecin, 9-nitrocamptothecin, taxanes, geldanamycin,ansamycins, and epothilones. In a more preferred embodiment, thechemotherapeutic moiety is SN-38. Preferably, in the conjugates of thepreferred embodiments, the antibody links to at least onechemotherapeutic moiety; preferably 1 to about 12 chemotherapeuticmoieties; most preferably about 6 to about 12 chemotherapeutic moieties.

Furthermore, in a preferred embodiment, the linker component 12′comprises a thiol group that reacts with a thiol-reactive residueintroduced at one or more lysine side chain amino groups of saidantibody. In such cases, the antibody is pre-derivatized with athiol-reactive group such as a maleimide, vinylsulfone, bromoacetamide,or iodoacetamide by procedures well described in the art.

In the context of this work, a process was surprisingly discovered bywhich CPT drug-linkers can be prepared wherein CPT additionally has a10-hydroxyl group. This process involves, but is not limited to, theprotection of the 10-hydroxyl group as a t-butyloxycarbonyl (BOC)derivative, followed by the preparation of the penultimate intermediateof the drug-linker conjugate. Usually, removal of BOC group requirestreatment with strong acid such as trifluoroacetic acid (TFA). Underthese conditions, the CPT 20-O-linker carbonate, containing protectinggroups to be removed, is also susceptible to cleavage, thereby givingrise to unmodified CPT. In fact, the rationale for using a mildlyremovable methoxytrityl (MMT) protecting group for the lysine side chainof the linker molecule, as enunciated in the art, was precisely to avoidthis possibility (Walker et al., 2002). It was discovered that selectiveremoval of phenolic BOC protecting group is possible by carrying outreactions for short durations, optimally 3-to-5 minutes. Under theseconditions, the predominant product was that in which the ‘BOC’ at10-hydroxyl position was removed, while the carbonate at ‘20’ positionwas intact.

An alternative approach involves protecting the CPT analog's 10-hydroxyposition with a group other than ‘BOC’, such that the the final productis ready for conjugation to antibodies without a need for deprotectingthe 10-OH protecting group. The 10-hydroxy protecting group, whichconverts the 10-OH into a phenolic carbonate or a phenolic ester, isreadily deprotected by physiological pH conditions or by esterases afterin vivo administration of the conjugate. The faster removal of aphenolic carbonate at the 10 position vs. a tertiary carbonate at the 20position of 10-hydroxycamptothecin under physiological condition hasbeen described by He et al. (He et al., Bioorganic & Medicinal Chemistry12: 4003-4008 (2004)). A 10-hydroxy protecting group on SN-38 can be‘COR’ where R can be a substituted alkyl such as “N(CH₃)₂—(CH₂)_(n)—”where n is 1-10 and wherein the terminal amino group is optionally inthe form of a quaternary salt for enhanced aqueous solubility, or asimple alkyl residue such as “CH₃—(CH₂)_(n)—” where n is 0-10, or it canbe an alkoxy moiety such as “CH₃—(CH₂)_(n)—O—” where n is 0-10, or“N(CH₃)₂—(CH₂)_(n)—O—” where n is 2-10, or“R₁O—(CH₂—CH₂O)_(n)—CH₂—CH₂—O—” where R₁ is ethyl or methyl and n is aninteger with values of 0-10. These 10-hydroxy derivatives are readilyprepared by treatment with the chloroformate of the chosen reagent, ifthe final derivative is to be a carbonate. Typically, the10-hydroxy-containing camptothecin such as SN-38 is treated with a molarequivalent of the chloroformate in dimethylformamide using triethylamineas the base. Under these conditions, the 20-OH position is unaffected.For forming 10-O-esters, the acid chloride of the chosen reagent isused.

In a preferred process of the preparation of a conjugate of a drugderivative and an antibody of the general formula 2, wherein thedescriptors L2, L1, AA and A-X are as described in earlier sections, thebifunctional drug moiety, [L2]-[L1]-[AA]_(m)-[A-X]-Drug is firstprepared, followed by the conjugation of the bifunctional drug moiety tothe antibody (indicated herein as “MAb”).

In a preferred process of the preparation of a conjugate of a drugderivative and an antibody of the general formula 2, wherein thedescriptors L2, L1, AA and A-OH are as described in earlier sections,the bifunctional drug moiety is prepared by first linking A-OH to theC-terminus of AA via an amide bond, followed by coupling the amine endof AA to a carboxylic acid group of L1. If AA is absent (i.e. m=0), A-OHis directly attached to L1 via an amide bond. The cross-linker,[L1]-[AA]_(m)-[A-OH], is attached to drug's hydroxyl or amino group, andthis is followed by attachment to the L1 moiety, by taking recourse tothe reaction between azide (or acetylene) and acetylene (or azide)groups in L1 and L2 via click chemistry.

In one embodiment, the antibody is a monoclonal antibody (MAb). In otherembodiments, the antibody may be a multivalent and/or multispecific MAb.The antibody may be a murine, chimeric, humanized, or human monoclonalantibody, and said antibody may be in intact, fragment (Fab, Fab′,F(ab)₂, F(ab′)₂), or sub-fragment (single-chain constructs) form, or ofan IgG1, IgG2a, IgG3, IgG4, IgA isotype, or submolecules therefrom.

In a preferred embodiment, the antibody binds to an antigen or epitopeof an antigen expressed on a cancer or malignant cell. The cancer cellis preferably a cell from a hematopoietic tumor, carcinoma, sarcoma,melanoma or a glial tumor. A preferred malignancy to be treatedaccording to the present invention is a malignant solid tumor orhematopoietic neoplasm.

In a preferred embodiment, the intracellularly-cleavable moiety may becleaved after it is internalized into the cell upon binding by theMAb-drug conjugate to a receptor thereof.

General Antibody Techniques

Techniques for preparing monoclonal antibodies against virtually anytarget antigen are well known in the art. See, for example, Köhler andMilstein, Nature 256: 495 (1975), and Coligan et al. (eds.), CURRENTPROTOCOLS IN IMMUNOLOGY, VOL. 1, pages 2.5.1-2.6.7 (John Wiley & Sons1991). Briefly, monoclonal antibodies can be obtained by injecting micewith a composition comprising an antigen, removing the spleen to obtainB-lymphocytes, fusing the B-lymphocytes with myeloma cells to producehybridomas, cloning the hybridomas, selecting positive clones whichproduce antibodies to the antigen, culturing the clones that produceantibodies to the antigen, and isolating the antibodies from thehybridoma cultures. The person of ordinary skill will realize that whereantibodies are to be administered to human subjects, the antibodies willbind to human antigens.

MAbs can be isolated and purified from hybridoma cultures by a varietyof well-established techniques. Such isolation techniques includeaffinity chromatography with Protein-A or Protein-G Sepharose,size-exclusion chromatography, and ion-exchange chromatography. See, forexample, Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3. Also, seeBaines et al., “Purification of Immunoglobulin G (IgG),” in METHODS INMOLECULAR BIOLOGY, VOL. 10, pages 79-104 (The Humana Press, Inc. 1992).

After the initial raising of antibodies to the immunogen, the antibodiescan be sequenced and subsequently prepared by recombinant techniques.Humanization and chimerization of murine antibodies and antibodyfragments are well known to those skilled in the art, as discussedbelow.

The skilled artisan will realize that the claimed methods andcompositions may utilize any of a wide variety of antibodies known inthe art. Antibodies of use may be commercially obtained from a widevariety of known sources. For example, a variety of antibody secretinghybridoma lines are available from the American Type Culture Collection(ATCC, Manassas, Va.). A large number of antibodies against variousdisease targets, including but not limited to tumor-associated antigens,have been deposited at the ATCC and/or have published variable regionsequences and are available for use in the claimed methods andcompositions. See, e.g., U.S. Pat. Nos. 7,312,318; 7,282,567; 7,151,164;7,074,403; 7,060,802; 7,056,509; 7,049,060; 7,045,132; 7,041,803;7,041,802; 7,041,293; 7,038,018; 7,037,498; 7,012,133; 7,001,598;6,998,468; 6,994,976; 6,994,852; 6,989,241; 6,974,863; 6,965,018;6,964,854; 6,962,981; 6,962,813; 6,956,107; 6,951,924; 6,949,244;6,946,129; 6,943,020; 6,939,547; 6,921,645; 6,921,645; 6,921,533;6,919,433; 6,919,078; 6,916,475; 6,905,681; 6,899,879; 6,893,625;6,887,468; 6,887,466; 6,884,594; 6,881,405; 6,878,812; 6,875,580;6,872,568; 6,867,006; 6,864,062; 6,861,511; 6,861,227; 6,861,226;6,838,282; 6,835,549; 6,835,370; 6,824,780; 6,824,778; 6,812,206;6,793,924; 6,783,758; 6,770,450; 6,767,711; 6,764,688; 6,764,681;6,764,679; 6,743,898; 6,733,981; 6,730,307; 6,720,155; 6,716,966;6,709,653; 6,693,176; 6,692,908; 6,689,607; 6,689,362; 6,689,355;6,682,737; 6,682,736; 6,682,734; 6,673,344; 6,653,104; 6,652,852;6,635,482; 6,630,144; 6,610,833; 6,610,294; 6,605,441; 6,605,279;6,596,852; 6,592,868; 6,576,745; 6,572,856; 6,566,076; 6,562,618;6,545,130; 6,544,749; 6,534,058; 6,528,625; 6,528,269; 6,521,227;6,518,404; 6,511,665; 6,491,915; 6,488,930; 6,482,598; 6,482,408;6,479,247; 6,468,531; 6,468,529; 6,465,173; 6,461,823; 6,458,356;6,455,044; 6,455,040, 6,451,310; 6,444,206; 6,441,143; 6,432,404;6,432,402; 6,419,928; 6,413,726; 6,406,694; 6,403,770; 6,403,091;6,395,276; 6,395,274; 6,387,350; 6,383,759; 6,383,484; 6,376,654;6,372,215; 6,359,126; 6,355,481; 6,355,444; 6,355,245; 6,355,244;6,346,246; 6,344,198; 6,340,571; 6,340,459; 6,331,175; 6,306,393;6,254,868; 6,187,287; 6,183,744; 6,129,914; 6,120,767; 6,096,289;6,077,499; 5,922,302; 5,874,540; 5,814,440; 5,798,229; 5,789,554;5,776,456; 5,736,119; 5,716,595; 5,677,136; 5,587,459; 5,443,953,5,525,338, the Examples section of each of which is incorporated hereinby reference. These are exemplary only and a wide variety of otherantibodies and their hybridomas are known in the art. The skilledartisan will realize that antibody sequences or antibody-secretinghybridomas against almost any disease-associated antigen may be obtainedby a simple search of the ATCC, NCBI and/or USPTO databases forantibodies against a selected disease-associated target of interest. Theantigen binding domains of the cloned antibodies may be amplified,excised, ligated into an expression vector, transfected into an adaptedhost cell and used for protein production, using standard techniqueswell known in the art. Isolated antibodies may be conjugated totherapeutic agents, such as camptothecins, using the techniquesdisclosed herein.

Chimeric and Humanized Antibodies

A chimeric antibody is a recombinant protein in which the variableregions of a human antibody have been replaced by the variable regionsof, for example, a mouse antibody, including thecomplementarity-determining regions (CDRs) of the mouse antibody.Chimeric antibodies exhibit decreased immunogenicity and increasedstability when administered to a subject. Methods for constructingchimeric antibodies are well known in the art (e.g., Leung et al., 1994,Hybridoma 13:469).

A chimeric monoclonal antibody may be humanized by transferring themouse CDRs from the heavy and light variable chains of the mouseimmunoglobulin into the corresponding variable domains of a humanantibody. The mouse framework regions (FR) in the chimeric monoclonalantibody are also replaced with human FR sequences. To preserve thestability and antigen specificity of the humanized monoclonal, one ormore human FR residues may be replaced by the mouse counterpartresidues. Humanized monoclonal antibodies may be used for therapeutictreatment of subjects. Techniques for production of humanized monoclonalantibodies are well known in the art. (See, e.g., Jones et al., 1986,Nature, 321:522; Riechmann et al., Nature, 1988, 332:323; Verhoeyen etal., 1988, Science, 239:1534; Carter et al., 1992, Proc. Nat'l Acad.Sci. USA, 89:4285; Sandhu, Crit. Rev. Biotech., 1992, 12:437; Tempest etal., 1991, Biotechnology 9:266; Singer et al., J. Immun., 1993,150:2844.)

Other embodiments may concern non-human primate antibodies. Generaltechniques for raising therapeutically useful antibodies in baboons maybe found, for example, in Goldenberg et al., WO 91/11465 (1991), and inLosman et al., Int. J. Cancer 46: 310 (1990). In another embodiment, anantibody may be a human monoclonal antibody. Such antibodies may beobtained from transgenic mice that have been engineered to producespecific human antibodies in response to antigenic challenge, asdiscussed below.

Human Antibodies

Methods for producing fully human antibodies using either combinatorialapproaches or transgenic animals transformed with human immunoglobulinloci are known in the art (e.g., Mancini et al., 2004, New Microbiol.27:315-28; Conrad and Scheller, 2005, Comb. Chem. High ThroughputScreen. 8:117-26; Brekke and Loset, 2003, Curr. Opin. Phamacol.3:544-50; each incorporated herein by reference). Such fully humanantibodies are expected to exhibit even fewer side effects than chimericor humanized antibodies and to function in vivo as essentiallyendogenous human antibodies. In certain embodiments, the claimed methodsand procedures may utilize human antibodies produced by such techniques.

In one alternative, the phage display technique may be used to generatehuman antibodies (e.g., Dantas-Barbosa et al., 2005, Genet. Mol. Res.4:126-40, incorporated herein by reference). Human antibodies may begenerated from normal humans or from humans that exhibit a particulardisease state, such as cancer (Dantas-Barbosa et al., 2005). Theadvantage to constructing human antibodies from a diseased individual isthat the circulating antibody repertoire may be biased towardsantibodies against disease-associated antigens.

In one non-limiting example of this methodology, Dantas-Barbosa et al.(2005) constructed a phage display library of human Fab antibodyfragments from osteosarcoma patients. Generally, total RNA was obtainedfrom circulating blood lymphocytes (Id.) Recombinant Fab were clonedfrom the μ, γ and κ chain antibody repertoires and inserted into a phagedisplay library (Id.) RNAs were converted to cDNAs and used to make FabcDNA libraries using specific primers against the heavy and light chainimmunoglobulin sequences (Marks et al., 1991, J Mol. Biol. 222:581-97,incorporated herein by reference). Library construction was performedaccording to Andris-Widhopf et al. (2000, In: Phage Display LaboratoryManual, Barbas et al. (eds), 1^(st) edition, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. pp. 9.1 to 9.22, incorporatedherein by reference). The final Fab fragments were digested withrestriction endonucleases and inserted into the bacteriophage genome tomake the phage display library. Such libraries may be screened bystandard phage display methods. The skilled artisan will realize thatthis technique is exemplary only and any known method for making andscreening human antibodies or antibody fragments by phage display may beutilized.

In another alternative, transgenic animals that have been geneticallyengineered to produce human antibodies may be used to generateantibodies against essentially any immunogenic target, using standardimmunization protocols as discussed above. Methods for obtaining humanantibodies from transgenic mice are described by Green et al., NatureGenet. 7:13 (1994), Lonberg et al., Nature 368:856 (1994), and Taylor etal., Int. Immun. 6:579 (1994). A non-limiting example of such a systemis the XENOMOUSE® (e.g., Green et al., 1999, J. Immunol. Methods231:11-23, incorporated herein by reference) from Abgenix (Fremont,Calif.), in which the mouse antibody genes have been inactivated andreplaced by functional human antibody genes, while the remainder of themouse immune system remains intact.

The transgenic mice were transformed with germline-configured YACs(yeast artificial chromosomes) that contained portions of the human IgHand Ig kappa loci, including the majority of the variable regionsequences, along accessory genes and regulatory sequences. The humanvariable region repertoire may be used to generate antibody producing Bcells, which may be processed into hybridomas by known techniques. AXENOMOUSE® immunized with a target antigen will produce human antibodiesby the normal immune response, which may be harvested and/or produced bystandard techniques discussed above. A variety of strains of geneticallyengineered mice are available, each of which is capable of producing adifferent class of antibody. Transgenically produced human antibodieshave been shown to have therapeutic potential, while retaining thepharmacokinetic properties of normal human antibodies (Green et al.,1999). The skilled artisan will realize that the claimed compositionsand methods are not limited to use of the XENOMOUSE® system but mayutilize any transgenic animal that has been genetically engineered toproduce human antibodies.

Production of Antibody Fragments

Some embodiments of the claimed methods and/or compositions may concernantibody fragments. Such antibody fragments may be obtained, forexample, by pepsin or papain digestion of whole antibodies byconventional methods. For example, antibody fragments may be produced byenzymatic cleavage of antibodies with pepsin to provide a 5S fragmentdenoted F(ab′)2. This fragment may be further cleaved using a thiolreducing agent and, optionally, a blocking group for the sulfhydrylgroups resulting from cleavage of disulfide linkages, to produce 3.5SFab′ monovalent fragments. Alternatively, an enzymatic cleavage usingpepsin produces two monovalent Fab fragments and an Fc fragment.Exemplary methods for producing antibody fragments are disclosed in U.S.Pat. Nos. 4,036,945; 4,331,647; Nisonoff et al., 1960, Arch. Biochem.Biophys., 89:230; Porter, 1959, Biochem. J., 73:119; Edelman et al.,1967, METHODS IN ENZYMOLOGY, page 422 (Academic Press), and Coligan etal. (eds.), 1991, CURRENT PROTOCOLS IN IMMUNOLOGY, (John Wiley & Sons).

Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments or other enzymatic, chemical or genetic techniques also may beused, so long as the fragments bind to the antigen that is recognized bythe intact antibody. For example, Fv fragments comprise an associationof V_(H) and V_(L) chains. This association can be noncovalent, asdescribed in Inbar et al., 1972, Proc. Nat'l. Acad. Sci. USA, 69:2659.Alternatively, the variable chains may be linked by an intermoleculardisulfide bond or cross-linked by chemicals such as glutaraldehyde. SeeSandhu, 1992, Crit. Rev. Biotech., 12:437.

Preferably, the Fv fragments comprise V_(H) and V_(L) chains connectedby a peptide linker. These single-chain antigen binding proteins (scFv)are prepared by constructing a structural gene comprising DNA sequencesencoding the V_(H) and V_(L) domains, connected by an oligonucleotideslinker sequence. The structural gene is inserted into an expressionvector that is subsequently introduced into a host cell, such as E.coli. The recombinant host cells synthesize a single polypeptide chainwith a linker peptide bridging the two V domains. Methods for producingscFvs are well-known in the art. See Whitlow et al., 1991, Methods: ACompanion to Methods in Enzymology 2:97; Bird et al., 1988, Science,242:423; U.S. Pat. No. 4,946,778; Pack et al., 1993, Bio/Technology,11:1271, and Sandhu, 1992, Crit. Rev. Biotech., 12:437.

Another form of an antibody fragment is a single-domain antibody (dAb),sometimes referred to as a single chain antibody. Techniques forproducing single-domain antibodies are well known in the art (see, e.g.,Cossins et al., Protein Expression and Purification, 2007, 51:253-59;Shuntao et al., Molec Immunol 2006, 43:1912-19; Tanha et al., J. Biol.Chem. 2001, 276:24774-780). Other types of antibody fragments maycomprise one or more complementarity-determining regions (CDRs). CDRpeptides (“minimal recognition units”) can be obtained by constructinggenes encoding the CDR of an antibody of interest. Such genes areprepared, for example, by using the polymerase chain reaction tosynthesize the variable region from RNA of antibody-producing cells. SeeLarrick et al., 1991, Methods: A Companion to Methods in Enzymology2:106; Ritter et al. (eds.), 1995, MONOCLONAL ANTIBODIES: PRODUCTION,ENGINEERING AND CLINICAL APPLICATION, pages 166-179 (CambridgeUniversity Press); Birch et al., (eds.), 1995, MONOCLONAL ANTIBODIES:PRINCIPLES AND APPLICATIONS, pages 137-185 (Wiley-Liss, Inc.)

Antibody Variations

In certain embodiments, the sequences of antibodies, such as the Fcportions of antibodies, may be varied to optimize the physiologicalcharacteristics of the conjugates, such as the half-life in serum.Methods of substituting amino acid sequences in proteins are widelyknown in the art, such as by site-directed mutagenesis (e.g. Sambrook etal., Molecular Cloning, A laboratory manual, 2^(nd) Ed, 1989). Inpreferred embodiments, the variation may involve the addition or removalof one or more glycosylation sites in the Fc sequence (e.g., U.S. Pat.No. 6,254,868, the Examples section of which is incorporated herein byreference). In other preferred embodiments, specific amino acidsubstitutions in the Fc sequence may be made (e.g., Hornick et al.,2000, J Nucl Med 41:355-62; Hinton et al., 2006, J Immunol 176:346-56;Petkova et al. 2006, Int Immunol 18:1759-69; U.S. Pat. No. 7,217,797;each incorporated herein by reference).

Target Antigens and Exemplary Antibodies

In a preferred embodiment, antibodies are used that recognize and/orbind to antigens that are expressed at high levels on target cells andthat are expressed predominantly or exclusively on diseased cells versusnormal tissues. More preferably, the antibodies internalize rapidlyfollowing binding. An exemplary rapidly internalizing antibody is theLL1 (anti-CD74) antibody, with a rate of internalization ofapproximately 8×10⁶ antibody molecules per cell per day (e.g., Hansen etal., 1996, Biochem 1 320:293-300). Thus, a “rapidly internalizing”antibody may be one with an internalization rate of about 1×10⁶ to about1×10⁷ antibody molecules per cell per day. Antibodies of use in theclaimed compositions and methods may include MAbs with properties asrecited above. Exemplary antibodies of use for therapy of, for example,cancer include but are not limited to LL1 (anti-CD74), LL2 or RFB4(anti-CD22), veltuzumab (hA20, anti-CD20), rituxumab (anti-CD20),obinutuzumab (GA101, anti-CD20), lambrolizumab (anti-PD-1 receptor),nivolumab (anti-PD-1 receptor), ipilimumab (anti-CTLA-4), RS7(anti-epithelial glycoprotein-1 (EGP-1, also known as TROP-2)), PAM4 orKC4 (both anti-mucin), MN-14 (anti-carcinoembryonic antigen (CEA, alsoknown as CD66e or CEACAM5), MN-15 or MN-3 (anti-CEACAM6), Mu-9(anti-colon-specific antigen-p), Immu 31 (an anti-alpha-fetoprotein), R1(anti-IGF-1R), A19 (anti-CD19), TAG-72 (e.g., CC49), Tn, J591 or HuJ591(anti-PSMA (prostate-specific membrane antigen)), AB-PG1-XG1-026(anti-PSMA dimer), D2/B (anti-PSMA), G250 (an anti-carbonic anhydrase IXMAb), L243 (anti-HLA-DR) alemtuzumab (anti-CD52), bevacizumab(anti-VEGF), cetuximab (anti-EGFR), gemtuzumab (anti-CD33), ibritumomabtiuxetan (anti-CD20); panitumumab (anti-EGFR); tositumomab (anti-CD20);PAM4 (aka clivatuzumab, anti-mucin) and trastuzumab (anti-ErbB2). Suchantibodies are known in the art (e.g., U.S. Pat. Nos. 5,686,072;5,874,540; 6,107,090; 6,183,744; 6,306,393; 6,653,104; 6,730.300;6,899,864; 6,926,893; 6,962,702; 7,074,403; 7,230,084; 7,238,785;7,238,786; 7,256,004; 7,282,567; 7,300,655; 7,312,318; 7,585,491;7,612,180; 7,642,239; and U.S. Patent Application Publ. No. 20050271671;20060193865; 20060210475; 20070087001; the Examples section of eachincorporated herein by reference.) Specific known antibodies of useinclude hPAM4 (U.S. Pat. No. 7,282,567), hA20 (U.S. Pat. No. 7,251,164),hA19 (U.S. Pat. No. 7,109,304), hIMMU-31 (U.S. Pat. No. 7,300,655), hLL1(U.S. Pat. No. 7,312,318,), hLL2 (U.S. Pat. No. 7,074,403), hMu-9 (U.S.Pat. No. 7,387,773), hL243 (U.S. Pat. No. 7,612,180), hMN-14 (U.S. Pat.No. 6,676,924), hMN-15 (U.S. Pat. No. 7,541,440), hR1 (U.S. patentapplication Ser. No. 12/772,645), hRS7 (U.S. Pat. No. 7,238,785), hMN-3(U.S. Pat. No. 7,541,440), AB-PG1-XG1-026 (U.S. patent application Ser.No. 11/983,372, deposited as ATCC PTA-4405 and PTA-4406) and D2/B (WO2009/130575) the text of each recited patent or application isincorporated herein by reference with respect to the Figures andExamples sections. In a particularly preferred embodiment, the antibodyis hRS7.

Other useful antigens that may be targeted using the describedconjugates include carbonic anhydrase IX, B7, CCCL19, CCCL21, CSAp,HER-2/neu, BrE3, CD1, CD1a, CD2, CD3, CD4, CD5, CD8, CD11A, CD14, CD15,CD16, CD18, CD19, CD20 (e.g., C2B8, hA20, 1F5 MAbs), CD21, CD22, CD23,CD25, CD29, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD44, CD45,CD46, CD52, CD54, CD55, CD59, CD64, CD67, CD70, CD74, CD79a, CD80, CD83,CD95, CD126, CD133, CD138, CD147, CD154, CEACAM5, CEACAM6, CTLA-4,alpha-fetoprotein (AFP), VEGF (e.g., bevacizumab, fibronectin splicevariant), ED-B fibronectin (e.g., L19), EGP-1 (TROP-2), EGP-2 (e.g.,17-1A), EGF receptor (ErbB1) (e.g., cetuximab), ErbB2, ErbB3, Factor H,FHL-1, Flt-3, folate receptor, Ga 733, GRO-β, HMGB-1, hypoxia induciblefactor (HIF), HM1.24, HER-2/neu, insulin-like growth factor (ILGF),IFN-γ, IFN-α, IFN-β, IFN-k, IL-2R, IL-4R, IL-6R, IL-13R, IL-15R, IL-17R,IL-18R, IL-2, IL-6, IL-8, IL-12, IL-15, IL-17, IL-18, IL-25, IP-10,IGF-1R, Ia, HM1.24, gangliosides, HCG, the HLA-DR antigen to which L243binds, CD66 antigens, i.e., CD66a-d or a combination thereof, MAGE,mCRP, MCP-1, MIP-1A, MIP-1B, macrophage migration-inhibitory factor(MIF), MUC1, MUC2, MUC3, MUC4, MUC5ac, placental growth factor (P1GF),PSA (prostate-specific antigen), PSMA, PAM4 antigen, PD-1 receptor,NCA-95, NCA-90, A3, A33, Ep-CAM, KS-1, Le(y), mesothelin, S100,tenascin, TAC, Tn antigen, Thomas-Friedenreich antigens, tumor necrosisantigens, tumor angiogenesis antigens, TNF-α, TRAIL receptor (R1 andR2), TROP-2, VEGFR, RANTES, T101, as well as cancer stem cell antigens,complement factors C3, C3a, C3b, C5a, C5, and an oncogene product.

A comprehensive analysis of suitable antigen (Cluster Designation, orCD) targets on hematopoietic malignant cells, as shown by flow cytometryand which can be a guide to selecting suitable antibodies fordrug-conjugated immunotherapy, is Craig and Foon, Blood prepublishedonline Jan. 15, 2008; DOL 10.1182/blood-2007-11-120535.

The CD66 antigens consist of five different glycoproteins with similarstructures, CD66a-e, encoded by the carcinoembryonic antigen (CEA) genefamily members, BCG, CGM6, NCA, CGM1 and CEA, respectively. These CD66antigens (e.g., CEACAM6) are expressed mainly in granulocytes, normalepithelial cells of the digestive tract and tumor cells of varioustissues. Also included as suitable targets for cancers are cancer testisantigens, such as NY-ESO-1 (Theurillat et al., Int. J. Cancer 2007;120(11):2411-7), as well as CD79a in myeloid leukemia (Kozlov et al.,Cancer Genet. Cytogenet. 2005; 163(1):62-7) and also B-cell diseases,and CD79b for non-Hodgkin's lymphoma (Poison et al., Blood110(2):616-623). A number of the aforementioned antigens are disclosedin U.S. Provisional Application Ser. No. 60/426,379, entitled “Use ofMulti-specific, Non-covalent Complexes for Targeted Delivery ofTherapeutics,” filed Nov. 15, 2002. Cancer stem cells, which areascribed to be more therapy-resistant precursor malignant cellpopulations (Hill and Penis, J. Natl. Cancer Inst. 2007; 99:1435-40),have antigens that can be targeted in certain cancer types, such asCD133 in prostate cancer (Maitland et al., Ernst Schering Found. Sympos.Proc. 2006; 5:155-79), non-small-cell lung cancer (Donnenberg et al., J.Control Release 2007; 122(3):385-91), and glioblastoma (Beier et al.,Cancer Res. 2007; 67(9):4010-5), and CD44 in colorectal cancer (Dalerbaer al., Proc. Natl. Acad. Sci. USA 2007; 104(24)10158-63), pancreaticcancer (Li et al., Cancer Res. 2007; 67(3):1030-7), and in head and necksquamous cell carcinoma (Prince et al., Proc. Natl. Acad. Sci. USA 2007;104(3)973-8). Another useful target for breast cancer therapy is theLIV-1 antigen described by Taylor et al. (Biochem. J. 2003; 375:51-9).The CD47 antigen is a further useful target for cancer stem cells (see,e.g., Naujokat et al., 2014, Immunotherapy 6:290-308; Goto et al., 2014,Eur J Cancer 50:1836-46; Unanue, 2013, Proc Natl Acad Sci USA110:10886-7).

For multiple myeloma therapy, suitable targeting antibodies have beendescribed against, for example, CD38 and CD138 (Stevenson, Mol Med 2006;12(11-12):345-346; Tassone et al., Blood 2004; 104(12):3688-96), CD74(Stein et al., ibid.), CS1 (Tai et al., Blood 2008; 112(4):1329-37, andCD40 (Tai et al., 2005; Cancer Res. 65(13):5898-5906).

Checkpoint inhibitor antibodies have been used in cancer therapy. Immunecheckpoints refer to inhibitory pathways in the immune system that areresponsible for maintaining self-tolerance and modulating the degree ofimmune system response to minimize peripheral tissue damage. However,tumor cells can also activate immune system checkpoints to decrease theeffectiveness of immune response against tumor tissues. Exemplarycheckpoint inhibitor antibodies against cytotoxic T-lymphocyte antigen 4(CTLA4, also known as CD152), programmed cell death protein 1 (PD1, alsoknown as CD279) and programmed cell death 1 ligand 1 (PD-L1, also knownas CD274), may be used in combination with one or more other agents toenhance the effectiveness of immune response against disease cells,tissues or pathogens. Exemplary anti-PD1 antibodies includelambrolizumab (MK-3475, MERCK), nivolumab (BMS-936558, BRISTOL-MYERSSQUIBB), AMP-224 (MERCK), and pidilizumab (CT-011, CURETECH LTD.).Anti-PD1 antibodies are commercially available, for example from ABCAM®(AB137132), BIOLEGEND® (EH12.2H7, RMP1-14) and AFFYMETRIX EBIOSCIENCE(J105, J116, MIH4). Exemplary anti-PD-L1 antibodies include MDX-1105(MEDAREX), MEDI4736 (MEDIMMUNE) MPDL3280A (GENENTECH) and BMS-936559(BRISTOL-MYERS SQUIBB). Anti-PD-L1 antibodies are also commerciallyavailable, for example from AFFYMETRIX EBIOSCIENCE (MIH1). Exemplaryanti-CTLA4 antibodies include ipilimumab (Bristol-Myers Squibb) andtremelimumab (PFIZER). Anti-PD1 antibodies are commercially available,for example from ABCAM® (AB134090), SINO BIOLOGICAL INC. (11159-H03H,11159-H08H), and THERMO SCIENTIFIC PIERCE (PAS-29572, PAS-23967,PAS-26465, MA1-12205, MA1-35914). Ipilimumab has recently received FDAapproval for treatment of metastatic melanoma (Wada et al., 2013, JTransl Med 11:89).

Macrophage migration inhibitory factor (MIF) is an important regulatorof innate and adaptive immunity and apoptosis. It has been reported thatCD74 is the endogenous receptor for MIF (Leng et al., 2003, J Exp Med197:1467-76). The therapeutic effect of antagonistic anti-CD74antibodies on MIF-mediated intracellular pathways may be of use fortreatment of a broad range of disease states, such as cancers of thebladder, prostate, breast, lung, colon and chronic lymphocytic leukemia(e.g., Meyer-Siegler et al., 2004, BMC Cancer 12:34; Shachar & Haran,2011, Leuk Lymphoma 52:1446-54); autoimmune diseases such as rheumatoidarthritis and systemic lupus erythematosus (Morand & Leech, 2005, FrontBiosci 10:12-22; Shachar & Haran, 2011, Leuk Lymphoma 52:1446-54);kidney diseases such as renal allograft rejection (Lan, 2008, NephronExp Nephrol. 109:e79-83); and numerous inflammatory diseases(Meyer-Siegler et al., 2009, Mediators Inflamm epub Mar. 22, 2009;Takahashi et al., 2009, Respir Res 10:33; Milatuzumab (hLL1) is anexemplary anti-CD74 antibody of therapeutic use for treatment ofMIF-mediated diseases.

Anti-TNF-α antibodies are known in the art and may be of use to treatimmune diseases, such as autoimmune disease, immune dysfunction (e.g.,graft-versus-host disease, organ transplant rejection) or diabetes.Known antibodies against TNF-α include the human antibody CDP571 (Ofeiet al., 2011, Diabetes 45:881-85); murine antibodies MTNFAI, M2TNFAI,M3TNFAI, M3TNFABI, M302B and M303 (Thermo Scientific, Rockford, Ill.);infliximab (Centocor, Malvern, Pa.); certolizumab pegol (UCB, Brussels,Belgium); and adalimumab (Abbott, Abbott Park, Ill.). These and manyother known anti-TNF-α antibodies may be used in the claimed methods andcompositions. Other antibodies of use for therapy of immunedysregulatory or autoimmune disease include, but are not limited to,anti-B-cell antibodies such as veltuzumab, epratuzumab, milatuzumab orhL243; tocilizumab (anti-IL-6 receptor); basiliximab (anti-CD25);daclizumab (anti-CD25); efalizumab (anti-CD11a); muromonab-CD3 (anti-CD3receptor); anti-CD40L (UCB, Brussels, Belgium); natalizumab (anti-a4integrin) and omalizumab (anti-IgE).

Type-1 and Type-2 diabetes may be treated using known antibodies againstB-cell antigens, such as CD22 (epratuzumab and hRFB4), CD74(milatuzumab), CD19 (hA19), CD20 (veltuzumab) or HLA-DR (hL243) (see,e.g., Winer et al., 2011, Nature Med 17:610-18). Anti-CD3 antibodiesalso have been proposed for therapy of type 1 diabetes (Cernea et al.,2010, Diabetes Metab Rev 26:602-05).

In another preferred embodiment, antibodies are used that internalizerapidly and are then re-expressed, processed and presented on cellsurfaces, enabling continual uptake and accretion of circulatingconjugate by the cell. An example of a most-preferred antibody/antigenpair is LL1, an anti-CD74 MAb (invariant chain, class II-specificchaperone, Ii) (see, e.g., U.S. Pat. Nos. 6,653,104; 7,312,318; theExamples section of each incorporated herein by reference). The CD74antigen is highly expressed on B-cell lymphomas (including multiplemyeloma) and leukemias, certain T-cell lymphomas, melanomas, colonic,lung, and renal cancers, glioblastomas, and certain other cancers (Onget al., Immunology 98:296-302 (1999)). A review of the use of CD74antibodies in cancer is contained in Stein et al., Clin Cancer Res. 2007Sep. 15; 13(18 Pt 2):55565-5563s, incorporated herein by reference.

The diseases that are preferably treated with anti-CD74 antibodiesinclude, but are not limited to, non-Hodgkin's lymphoma, Hodgkin'sdisease, melanoma, lung, renal, colonic cancers, glioblastomemultiforme, histiocytomas, myeloid leukemias, and multiple myeloma.Continual expression of the CD74 antigen for short periods of time onthe surface of target cells, followed by internalization of the antigen,and re-expression of the antigen, enables the targeting LL1 antibody tobe internalized along with any chemotherapeutic moiety it carries. Thisallows a high, and therapeutic, concentration of LL1-chemotherapeuticdrug conjugate to be accumulated inside such cells. InternalizedLL1-chemotherapeutic drug conjugates are cycled through lysosomes andendosomes, and the chemotherapeutic moiety is released in an active formwithin the target cells.

Antibodies of use to treat autoimmune disease or immune systemdysfunctions (e.g., graft-versus-host disease, organ transplantrejection) are known in the art and may be conjugated to SN-38 using thedisclosed methods and compositions. Antibodies of use to treatautoimmune/immune dysfunction disease may bind to exemplary antigensincluding, but not limited to, BCL-1, BCL-2, BCL-6, CD1a, CD2, CD3, CD4,CD5, CD7, CD8, CD10, CD11b, CD11c, CD13, CD14, CD15, CD16, CD19, CD20,CD21, CD22, CD23, CD25, CD33, CD34, CD38, CD40, CD40L, CD41a, CD43,CD45, CD55, TNF-alpha, interferon and HLA-DR. Antibodies that bind tothese and other target antigens, discussed above, may be used to treatautoimmune or immune dysfunction diseases. Autoimmune diseases that maybe treated with immunoconjugates may include acute idiopathicthrombocytopenic purpura, chronic idiopathic thrombocytopenic purpura,dermatomyositis, Sydenham's chorea, myasthenia gravis, systemic lupuserythematosus, lupus nephritis, rheumatic fever, polyglandularsyndromes, bullous pemphigoid, diabetes mellitus, Henoch-Schonleinpurpura, post-streptococcal nephritis, erythema nodosum, Takayasu'sarteritis, ANCA-associated vasculitides, Addison's disease, rheumatoidarthritis, multiple sclerosis, sarcoidosis, ulcerative colitis, erythemamultiforme, IgA nephropathy, polyarteritis nodosa, ankylosingspondylitis, Goodpasture's syndrome, thromboangitis obliterans,Sjögren's syndrome, primary biliary cirrhosis, Hashimoto's thyroiditis,thyrotoxicosis, scleroderma, chronic active hepatitis,polymyositis/dermatomyositis, polychondritis, bullous pemphigoid,pemphigus vulgaris, Wegener's granulomatosis, membranous nephropathy,amyotrophic lateral sclerosis, tabes dorsalis, giant cellarteritis/polymyalgia, pernicious anemia, rapidly progressiveglomerulonephritis, psoriasis or fibrosing alveolitis.

The antibodies discussed above and other known antibodies againstdisease-associated antigens may be used as CPT-conjugates, morepreferably SN-38-conjugates, in the practice of the claimed methods andcompositions. In a most preferred embodiment, the drug-conjugatedantibody is an anti-Trop-2-SN-38 (e.g., hRS7-SN-38) conjugate.

Bispecific and Multispecific Antibodies

Bispecific antibodies are useful in a number of biomedical applications.For instance, a bispecific antibody with binding sites for a tumor cellsurface antigen and for a T-cell surface receptor can direct the lysisof specific tumor cells by T cells. Bispecific antibodies recognizinggliomas and the CD3 epitope on T cells have been successfully used intreating brain tumors in human patients (Nitta, et al. Lancet. 1990;355:368-371). A preferred bispecific antibody is an anti-CD3 X anti-CD19antibody. In alternative embodiments, an anti-CD3 antibody or fragmentthereof may be attached to an antibody or fragment against anotherB-cell associated antigen, such as anti-CD3 X anti-Trop-2, anti-CD3 Xanti-CD20, anti-CD3 X anti-CD22, anti-CD3 X anti-HLA-DR or anti-CD3 Xanti-CD74. In certain embodiments, the techniques and compositions fortherapeutic agent conjugation disclosed herein may be used withbispecific or multispecific antibodies as the targeting moieties.

Numerous methods to produce bispecific or multispecific antibodies areknown, as disclosed, for example, in U.S. Pat. No. 7,405,320, theExamples section of which is incorporated herein by reference.Bispecific antibodies can be produced by the quadroma method, whichinvolves the fusion of two different hybridomas, each producing amonoclonal antibody recognizing a different antigenic site (Milstein andCuello, Nature, 1983; 305:537-540).

Another method for producing bispecific antibodies usesheterobifunctional cross-linkers to chemically tether two differentmonoclonal antibodies (Staerz, et al. Nature, 1985; 314:628-631; Perez,et al. Nature, 1985; 316:354-356). Bispecific antibodies can also beproduced by reduction of each of two parental monoclonal antibodies tothe respective half molecules, which are then mixed and allowed toreoxidize to obtain the hybrid structure (Staerz and Bevan. Proc NatlAcad Sci USA. 1986; 83:1453-1457). Another alternative involveschemically cross-linking two or three separately purified Fab′ fragmentsusing appropriate linkers. (See, e.g., European Patent Application0453082).

Other methods include improving the efficiency of generating hybridhybridomas by gene transfer of distinct selectable markers viaretrovirus-derived shuttle vectors into respective parental hybridomas,which are fused subsequently (DeMonte, et al. Proc Natl Acad Sci USA.1990, 87:2941-2945); or transfection of a hybridoma cell line withexpression plasmids containing the heavy and light chain genes of adifferent antibody.

Cognate V_(H) and V_(L) domains can be joined with a peptide linker ofappropriate composition and length (usually consisting of more than 12amino acid residues) to form a single-chain Fv (scFv) with bindingactivity. Methods of manufacturing scFvs are disclosed in U.S. Pat. Nos.4,946,778 and 5,132,405, the Examples section of each of which isincorporated herein by reference. Reduction of the peptide linker lengthto less than 12 amino acid residues prevents pairing of V_(H) and V_(L)domains on the same chain and forces pairing of V_(H) and V_(L) domainswith complementary domains on other chains, resulting in the formationof functional multimers. Polypeptide chains of V_(H) and V_(L) domainsthat are joined with linkers between 3 and 12 amino acid residues formpredominantly dimers (termed diabodies). With linkers between 0 and 2amino acid residues, trimers (termed triabody) and tetramers (termedtetrabody) are favored, but the exact patterns of oligomerization appearto depend on the composition as well as the orientation of V-domains(V_(H)-linker-V_(L) or V_(L)-linker-V_(H)), in addition to the linkerlength.

These techniques for producing multispecific or bispecific antibodiesexhibit various difficulties in terms of low yield, necessity forpurification, low stability or the labor-intensiveness of the technique.More recently, a technique known as “dock and lock” (DML®) has beenutilized to produce combinations of virtually any desired antibodies,antibody fragments and other effector molecules (see, e.g., U.S. Pat.Nos. 7,521,056; 7,527,787; 7,534,866; 7,550,143; 7,666,400; 7,858,070;7,871,622; 7,906,121; 7,906,118; 8,163,291; 7,901,680; 7,981,398;8,003,111 and 8,034,352, the Examples section of each of whichincorporated herein by reference). The technique utilizes complementaryprotein binding domains, referred to as anchoring domains (AD) anddimerization and docking domains (DDD), which bind to each other andallow the assembly of complex structures, ranging from dimers, trimers,tetramers, quintamers and hexamers. These form stable complexes in highyield without requirement for extensive purification. The DNL® techniqueallows the assembly of monospecific, bispecific or multispecificantibodies. Any of the techniques known in the art for making bispecificor multispecific antibodies may be utilized in the practice of thepresently claimed methods.

In various embodiments, a conjugate as disclosed herein may be part of acomposite, multispecific antibody. Such antibodies may contain two ormore different antigen binding sites, with differing specificities. Themultispecific composite may bind to different epitopes of the sameantigen, or alternatively may bind to two different antigens. Some ofthe more preferred target combinations include those listed in Table 1.This is a list of examples of preferred combinations, but is notintended to be exhaustive.

TABLE 1 Some Examples of multispecific antibodies. First target Secondtarget MIF A second proinflammatory effector cytokine, especiallyHMGB-1, TNF-α, IL-1, or IL-6 MIF Proinflammatory effector chemokine,especially MCP-1, RANTES, MIP- 1A, or MIP-1B MIF Proinflammatoryeffector receptor, especially IL-6R, IL-13R, and IL-15R MIF Coagulationfactor, especially TF or thrombin MIF Complement factor, especially C3,C5, C3a, or C5a MIF Complement regulatory protein, especially CD46,CD55, CD59, and mCRP MIF Cancer associated antigen or receptor HMGB-1 Asecond proinflammatory effector cytokine, especially MIF, TNF-α, IL-1,or IL-6 HMGB-1 Proinflammatory effector chemokine, especially MCP-1,RANTES, MIP- 1A, or MIP-1B HMGB-1 Proinflammatory effector receptorespecially MCP-1, RANTES, MIP-1A, or MIP-1B HMGB-1 Coagulation factor,especially TF or thrombin HMGB-1 Complement factor, especially C3, C5,C3a, or C5a HMGB-1 Complement regulatory protein, especially CD46, CD55,CD59, and mCRP HMGB-1 Cancer associated antigen or receptor TNF-α Asecond proinflammatory effector cytokine, especially MIF, HMGB-1, TNF-α,IL-1, or IL-6 TNF-α Proinflammatory effector chemokine, especiallyMCP-1, RANTES, MIP- 1A, or MIP-1B TNF-α Proinflammatory effectorreceptor, especially IL-6R IL-13R, and IL-15R TNF-α Coagulation factor,especially TF or thrombin TNF-α Complement factor, especially C3, C5,C3a, or C5a TNF-α Complement regulatory protein, especially CD46, CD55,CD59, and mCRP TNF-α Cancer associated antigen or receptor LPSProinflammatory effector cytokine, especially MIF, HMGB-1, TNF-α, IL-1,or IL-6 LPS Proinflammatory effector chemokine, especially MCP-1,RANTES, MIP- 1A, or MIP-1B LPS Proinflammatory effector receptor,especially IL-6R IL-13R, and IL-15R LPS Coagulation factor, especiallyTF or thrombin LPS Complement factor, especially C3, C5, C3a, or C5a LPSComplement regulatory protein, especially CD46, CD55, CD59, and mCRP TFor thrombin Proinflammatory effector cytokine, especially MIF, HMGB-1,TNF-α, IL-1, or IL-6 TF or thrombin Proinflammatory effector chemokine,especially MCP-1, RANTES, MIP- 1A, or MIP-1B TF or thrombinProinflammatory effector receptor, especially IL-6R IL-13R, and IL-15RTF or thrombin Complement factor, especially C3, C5, C3a, or C5a TF orthrombin Complement regulatory protein, especially CD46, CD55, CD59, andmCRP TF or thrombin Cancer associated antigen or receptor

Still other combinations, such as are preferred for cancer therapies,include CD20+CD22 antibodies, CD74+CD20 antibodies, CD74+CD22antibodies, CEACAM5 (CEA)+CEACAM6 (NCA) antibodies, insulin-like growthfactor (ILGF)+CEACAM5 antibodies, EGP-1 (e.g., RS-7)+ILGF antibodies,CEACAM5+EGFR antibodies, IL6+CEACAM6 antibodies. Such antibodies neednot only be used in combination, but can be combined as fusion proteinsof various forms, such as IgG, Fab, scFv, and the like, as described inU.S. Pat. Nos. 6,083,477; 6,183,744 and 6,962,702 and U.S. PatentApplication Publication Nos. 20030124058; 20030219433; 20040001825;20040202666; 20040219156; 20040219203; 20040235065; 20050002945;20050014207; 20050025709; 20050079184; 20050169926; 20050175582;20050249738; 20060014245 and 20060034759, the Examples section of eachincorporated herein by reference.

DOCK-AND-LOCK® (DNL®)

In preferred embodiments, a bivalent or multivalent antibody is formedas a DOCK-AND-LOCK® (DNL®) complex (see, e.g., U.S. Pat. Nos. 7,521,056;7,527,787; 7,534,866; 7,550,143; 7,666,400; 7,858,070; 7,871,622;7,906,121; 7,906,118; 8,163,291; 7,901,680; 7,981,398; 8,003,111 and8,034,352, the Examples section of each of which is incorporated hereinby reference.) Generally, the technique takes advantage of the specificand high-affinity binding interactions that occur between a dimerizationand docking domain (DDD) sequence of the regulatory (R) subunits ofcAMP-dependent protein kinase (PKA) and an anchor domain (AD) sequencederived from any of a variety of AKAP proteins (Baillie et al., FEBSLetters. 2005; 579: 3264. Wong and Scott, Nat. Rev. Mol. Cell Biol.2004; 5: 959). The DDD and AD peptides may be attached to any protein,peptide or other molecule. Because the DDD sequences spontaneouslydimerize and bind to the AD sequence, the technique allows the formationof complexes between any selected molecules that may be attached to DDDor AD sequences.

Although the standard DNL® complex comprises a trimer with twoDDD-linked molecules attached to one AD-linked molecule, variations incomplex structure allow the formation of dimers, trimers, tetramers,pentamers, hexamers and other multimers. In some embodiments, the DNL®complex may comprise two or more antibodies, antibody fragments orfusion proteins which bind to the same antigenic determinant or to twoor more different antigens. The DNL® complex may also comprise one ormore other effectors, such as proteins, peptides, immunomodulators,cytokines, interleukins, interferons, binding proteins, peptide ligands,carrier proteins, toxins, ribonucleases such as onconase, inhibitoryoligonucleotides such as siRNA, antigens or xenoantigens, polymers suchas PEG, enzymes, therapeutic agents, hormones, cytotoxic agents,anti-angiogenic agents, pro-apoptotic agents or any other molecule oraggregate.

PKA, which plays a central role in one of the best studied signaltransduction pathways triggered by the binding of the second messengercAMP to the R subunits, was first isolated from rabbit skeletal musclein 1968 (Walsh et al., J. Biol. Chem. 1968; 243:3763). The structure ofthe holoenzyme consists of two catalytic subunits held in an inactiveform by the R subunits (Taylor, J. Biol. Chem. 1989; 264:8443). Isozymesof PKA are found with two types of R subunits (RI and RII), and eachtype has α and β isoforms (Scott, Pharmacol. Ther. 1991; 50:123). Thus,the four isoforms of PKA regulatory subunits are RIα, RIβ, RIIα andRIIβ. The R subunits have been isolated only as stable dimers and thedimerization domain has been shown to consist of the first 44amino-terminal residues of RIIα (Newlon et al., Nat. Struct. Biol. 1999;6:222). As discussed below, similar portions of the amino acid sequencesof other regulatory subunits are involved in dimerization and docking,each located near the N-terminal end of the regulatory subunit. Bindingof cAMP to the R subunits leads to the release of active catalyticsubunits for a broad spectrum of serine/threonine kinase activities,which are oriented toward selected substrates through thecompartmentalization of PKA via its docking with AKAPs (Scott et al., J.Biol. Chem. 1990; 265; 21561)

Since the first AKAP, microtubule-associated protein-2, wascharacterized in 1984 (Lohmann et al., Proc. Natl. Acad. Sci USA. 1984;81:6723), more than 50 AKAPs that localize to various sub-cellularsites, including plasma membrane, actin cytoskeleton, nucleus,mitochondria, and endoplasmic reticulum, have been identified withdiverse structures in species ranging from yeast to humans (Wong andScott, Nat. Rev. Mol. Cell Biol. 2004; 5:959). The AD of AKAPs for PKAis an amphipathic helix of 14-18 residues (Carr et al., J. Biol. Chem.1991; 266:14188). The amino acid sequences of the AD are quite variedamong individual AKAPs, with the binding affinities reported for RHdimers ranging from 2 to 90 nM (Alto et al., Proc. Natl. Acad. Sci. USA.2003; 100:4445). AKAPs will only bind to dimeric R subunits. For humanRIIα, the AD binds to a hydrophobic surface formed by the 23amino-terminal residues (Colledge and Scott, Trends Cell Biol. 1999;6:216). Thus, the dimerization domain and AKAP binding domain of humanRIIα are both located within the same N-terminal 44 amino acid sequence(Newlon et al., Nat. Struct. Biol. 1999; 6:222; Newlon et al., EMBO J.2001; 20:1651), which is termed the DDD herein.

We have developed a platform technology to utilize the DDD of human PKAregulatory subunits and the AD of AKAP as an excellent pair of linkermodules for docking any two entities, referred to hereafter as A and B,into a noncovalent complex, which could be further locked into a DNL®complex through the introduction of cysteine residues into both the DDDand AD at strategic positions to facilitate the formation of disulfidebonds. The general methodology of the approach is as follows. Entity Ais constructed by linking a DDD sequence to a precursor of A, resultingin a first component hereafter referred to as a. Because the DDDsequence would effect the spontaneous formation of a dimer, A would thusbe composed of a₂. Entity B is constructed by linking an AD sequence toa precursor of B, resulting in a second component hereafter referred toas b. The dimeric motif of DDD contained in az will create a dockingsite for binding to the AD sequence contained in b, thus facilitating aready association of a₂ and b to form a binary, trimeric complexcomposed of a₂b. This binding event is made irreversible with asubsequent reaction to covalently secure the two entities via disulfidebridges, which occurs very efficiently based on the principle ofeffective local concentration because the initial binding interactionsshould bring the reactive thiol groups placed onto both the DDD and ADinto proximity (Chmura et al., Proc. Natl. Acad. Sci. USA. 2001;98:8480) to ligate site-specifically. Using various combinations oflinkers, adaptor modules and precursors, a wide variety of DNL®constructs of different stoichiometry may be produced and used (see,e.g., U.S. Nos. 7,550,143; 7,521,056; 7,534,866; 7,527,787 and7,666,400.)

By attaching the DDD and AD away from the functional groups of the twoprecursors, such site-specific ligations are also expected to preservethe original activities of the two precursors. This approach is modularin nature and potentially can be applied to link, site-specifically andcovalently, a wide range of substances, including peptides, proteins,antibodies, antibody fragments, and other effector moieties with a widerange of activities. Utilizing the fusion protein method of constructingAD and DDD conjugated effectors described in the Examples below,virtually any protein or peptide may be incorporated into a DNL®construct. However, the technique is not limiting and other methods ofconjugation may be utilized.

A variety of methods are known for making fusion proteins, includingnucleic acid synthesis, hybridization and/or amplification to produce asynthetic double-stranded nucleic acid encoding a fusion protein ofinterest. Such double-stranded nucleic acids may be inserted intoexpression vectors for fusion protein production by standard molecularbiology techniques (see, e.g. Sambrook et al., Molecular Cloning, Alaboratory manual, 2^(nd) Ed, 1989). In such preferred embodiments, theAD and/or DDD moiety may be attached to either the N-terminal orC-terminal end of an effector protein or peptide. However, the skilledartisan will realize that the site of attachment of an AD or DDD moietyto an effector moiety may vary, depending on the chemical nature of theeffector moiety and the part(s) of the effector moiety involved in itsphysiological activity. Site-specific attachment of a variety ofeffector moieties may be performed using techniques known in the art,such as the use of bivalent cross-linking reagents and/or other chemicalconjugation techniques.

In various embodiments, an antibody or antibody fragment may beincorporated into a DNL® complex by, for example, attaching a DDD or ADmoiety to the C-terminal end of the antibody heavy chain, as describedin detail below. In more preferred embodiments, the DDD or AD moiety,more preferably the AD moiety, may be attached to the C-terminal end ofthe antibody light chain (see, e.g., U.S. patent application Ser. No.13/901,737, filed May 24, 2013, the Examples section of which isincorporated herein by reference.)

Structure-Function Relationships in AD and DDD Moieties

For different types of DNL® constructs, different AD or DDD sequencesmay be utilized. Exemplary DDD and AD sequences are provided below.

DDD1 (SEQ ID NO: 1) SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA DDD2(SEQ ID NO: 2) CGHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA AD1(SEQ ID NO: 3) QIEYLAKQIVDNAIQQA AD2 (SEQ ID NO: 4)CGQIEYLAKQIVDNAIQQAGC

The skilled artisan will realize that DDD1 and DDD2 are based on the DDDsequence of the human RIIα isoform of protein kinase A. However, inalternative embodiments, the DDD and AD moieties may be based on the DDDsequence of the human Ma form of protein kinase A and a correspondingAKAP sequence, as exemplified in DDD3, DDD3C and AD3 below.

DDD3 (SEQ ID NO: 5) SLRECELYVQKHNIQALLKDSIVQLCTARPERPMAFLREYFERLEKEEAKDDD3C (SEQ ID NO: 6) MSCGGSLRECELYVQKHNIQALLKDSIVQLCTARPERPMAFLREYFERLEKEEAK AD3 (SEQ ID NO: 7) CGFEELAWKIAKMIWSDVFQQGC

In other alternative embodiments, other sequence variants of AD and/orDDD moieties may be utilized in construction of the DNL® complexes. Forexample, there are only four variants of human PKA DDD sequences,corresponding to the DDD moieties of PKA RIα, RIIα, RIβ and RIIβ. TheRIIα DDD sequence is the basis of DDD1 and DDD2 disclosed above. Thefour human PKA DDD sequences are shown below. The DDD sequencerepresents residues 1-44 of RIIα, 1-44 of RIIβ, 12-61 of RIα and 13-66of RIβ. (Note that the sequence of DDD1 is modified slightly from thehuman PKA RIIα DDD moiety.)

PKA RIα (SEQ ID NO: 8) SLRECELYVQKHNIQALLKDVSIVQLCTARPERPMAFLREYFEKLEKEEAK PKA RIβ (SEQ ID NO: 9)SLKGCELYVQLHGIQQVLKDCIVHLCISKPERPMKFLREHFEKLEKEEN RQILA PKA RIIα(SEQ ID NO: 10) SHIQIPPGLTELLQGYTVEVGQQPPDLVDFAVEYFTRLREARRQ PKA RIIβ(SEQ ID NO: 11) SIEIPAGLTELLQGFTVEVLRHQPADLLEFALQHFTRLQQENER

The structure-function relationships of the AD and DDD domains have beenthe subject of investigation. (See, e.g., Burns-Hamuro et al., 2005,Protein Sci 14:2982-92; Carr et al., 2001, J Boil Chem 276:17332-38;Alto et al., 2003, Proc Natl Acad Sci USA 100:4445-50; Hundsrucker etal., 2006, Biochem J 396:297-306; Stokka et al., 2006, Biochem J400:493-99; Gold et al., 2006, Mol Cell 24:383-95; Kinderman et al.,2006, Mol Cell 24:397-408, the entire text of each of which isincorporated herein by reference.)

For example, Kinderman et al. (2006, Mol Cell 24:397-408) examined thecrystal structure of the AD-DDD binding interaction and concluded thatthe human DDD sequence contained a number of conserved amino acidresidues that were important in either dimer formation or AKAP binding,underlined in SEQ ID NO:1 below. (See FIG. 1 of Kinderman et al., 2006,incorporated herein by reference.) The skilled artisan will realize thatin designing sequence variants of the DDD sequence, one would desirablyavoid changing any of the underlined residues, while conservative aminoacid substitutions might be made for residues that are less critical fordimerization and AKAP binding.

(SEQ ID NO: 1) SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA

As discussed in more detail below, conservative amino acid substitutionshave been characterized for each of the twenty common L-amino acids.Thus, based on the data of Kinderman (2006) and conservative amino acidsubstitutions, potential alternative DDD sequences based on SEQ ID NO:1are shown in Table 2. In devising Table 2, only highly conservativeamino acid substitutions were considered. For example, charged residueswere only substituted for residues of the same charge, residues withsmall side chains were substituted with residues of similar size,hydroxyl side chains were only substituted with other hydroxyls, etc.Because of the unique effect of proline on amino acid secondarystructure, no other residues were substituted for proline. The skilledartisan will realize that an almost unlimited number of alternativespecies within the genus of DDD moieties can be constructed by standardtechniques, for example using a commercial peptide synthesizer or wellknown site-directed mutagenesis techniques. The effect of the amino acidsubstitutions on AD moiety binding may also be readily determined bystandard binding assays, for example as disclosed in Alto et al. (2003,Proc Natl Acad Sci USA 100:4445-50).

TABLE 2Conservative Amino Acid Substitutions in DDD1 (SEQ ID NO: 1). Consensussequence disclosed as SEQ ID NO: 12. S H I Q I P P G L T E L L Q G Y T VE V L R T K N A S D N A S D K R Q Q P P D L V E F A V E Y F T R L R E AR A N N E D L D S K K D L K L I I I V V V

Alto et al. (2003, Proc Natl Acad Sci USA 100:4445-50) performed abioinformatic analysis of the AD sequence of various AKAP proteins todesign an RII selective AD sequence called AKAP-IS (SEQ ID NO:3), with abinding constant for DDD of 0.4 nM. The AKAP-IS sequence was designed asa peptide antagonist of AKAP binding to PKA. Residues in the AKAP-ISsequence where substitutions tended to decrease binding to DDD areunderlined in SEQ ID NO:3 below. The skilled artisan will realize thatin designing sequence variants of the AD sequence, one would desirablyavoid changing any of the underlined residues, while conservative aminoacid substitutions might be made for residues that are less critical forDDD binding. Table 3 shows potential conservative amino acidsubstitutions in the sequence of AKAP-IS (AD1, SEQ ID NO:3), similar tothat shown for DDD1 (SEQ ID NO:1) in Table 2 above.

Again, a very large number of species within the genus of possible ADmoiety sequences could be made, tested and used by the skilled artisan,based on the data of Alto et al. (2003). It is noted that FIG. 2 of Alto(2003) shows an even large number of potential amino acid substitutionsthat may be made, while retaining binding activity to DDD moieties,based on actual binding experiments.

AKAP-IS (SEQ ID NO: 3) QIEYLAKQIVDNAIQQA

TABLE 3Conservative Amino Acid Substitutions in AD1 (SEQ ID NO: 3). Consensussequence disclosed as SEQ ID NO: 13 Q I E Y L A K Q I V D N A I Q Q A NL D F I R N E Q N N L V T V I S V

Gold et al. (2006, Mol Cell 24:383-95) utilized crystallography andpeptide screening to develop a SuperAKAP-IS sequence (SEQ ID NO:14),exhibiting a five order of magnitude higher selectivity for the RIIisoform of PKA compared with the RI isoform. Underlined residuesindicate the positions of amino acid substitutions, relative to theAKAP-IS sequence, which increased binding to the DDD moiety of RIIα. Inthis sequence, the N-terminal Q residue is numbered as residue number 4and the C-terminal A residue is residue number 20. Residues wheresubstitutions could be made to affect the affinity for RIIα wereresidues 8, 11, 15, 16, 18, 19 and 20 (Gold et al., 2006). It iscontemplated that in certain alternative embodiments, the SuperAKAP-ISsequence may be substituted for the AKAP-IS AD moiety sequence toprepare DNL® constructs. Other alternative sequences that might besubstituted for the AKAP-IS AD sequence are shown in SEQ ID NO:14-17.Substitutions relative to the AKAP-IS sequence are underlined. It isanticipated that, as with the AD2 sequence shown in SEQ ID NO:4, the ADmoiety may also include the additional N-terminal residues cysteine andglycine and C-terminal residues glycine and cysteine.

SuperAKAP-IS (SEQ ID NO: 14) QIEYVAKQIVDYAIHQAAlternative AKAP sequences (SEQ ID NO: 15) QIEYKAKQIVDHAIHQA(SEQ ID NO: 16) QIEYHAKQIVDHAIHQA (SEQ ID NO: 17) QIEYVAKQIVDHAIHQA

FIG. 2 of Gold et al. disclosed additional DDD-binding sequences from avariety of AKAP proteins. Stokka et al. (2006, Biochem J 400:493-99)also developed peptide competitors of AKAP binding to PKA. The peptideantagonists were designated as Ht31, RIAD and PV-38. The Ht-31 peptideexhibited a greater affinity for the RH isoform of PKA, while the RIADand PV-38 showed higher affinity for RI.

Hundsrucker et al. (2006, Biochem J 396:297-306) developed still otherpeptide competitors for AKAP binding to PKA, with a binding constant aslow as 0.4 nM to the DDD of the RII form of PKA. The sequences ofvarious AKAP antagonistic peptides are provided in Table 1 ofHundsrucker et al. The AKAPIS represented a synthetic RIIsubunit-binding peptide. All other peptides are derived from theRII-binding domains of known AKAPs.

Residues that were highly conserved among the AD domains of differentAKAP proteins are indicated below by underlining with reference to theAKAP IS sequence (SEQ ID NO:3). The residues are the same as observed byAlto et al. (2003), with the addition of the C-terminal alanine residue.(See FIG. 4 of Hundsrucker et al. (2006), incorporated herein byreference.) The sequences of peptide antagonists with particularly highaffinities for the RII DDD sequence were those of AKAP-IS,AKAP7β-wt-pep, AKAP7β-L304T-pep and AKAP7β-L308D-pep.

AKAP-IS (SEQ ID NO: 3) QIEYLAKQIVDNAIQQA

Carr et al. (2001, J Biol Chem 276:17332-38) examined the degree ofsequence homology between different AKAP-binding DDD sequences fromhuman and non-human proteins and identified residues in the DDDsequences that appeared to be the most highly conserved among differentDDD moieties. These are indicated below by underlining with reference tothe human PKA RIIα DDD sequence of SEQ ID NO: 1. Residues that wereparticularly conserved are further indicated by italics. The residuesoverlap with, but are not identical to those suggested by Kinderman etal. (2006) to be important for binding to AKAP proteins. The skilledartisan will realize that in designing sequence variants of DDD, itwould be most preferred to avoid changing the most conserved residues(italicized), and it would be preferred to also avoid changing theconserved residues (underlined), while conservative amino acidsubstitutions may be considered for residues that are neither underlinednor italicized..

(SEQ ID NO: 1) SHIQ IP P GL TELLQGYT V EVLR Q QP P DLVEFA VE YF TR L REAR A

A modified set of conservative amino acid substitutions for the DDD1(SEQ ID NO:1) sequence, based on the data of Carr et al. (2001) is shownin Table 4. Even with this reduced set of substituted sequences, thereare numerous possible alternative DDD moiety sequences that may beproduced, tested and used by the skilled artisan without undueexperimentation. The skilled artisan could readily derive suchalternative DDD amino acid sequences as disclosed above for Table 2 andTable 3.

TABLE 4Conservative Amino Acid Substitutions in DDD1 (SEQ ID NO: 1). Consensussequence disclosed as SEQ ID NO: 18. S H I Q

P

T E

Q

V

T N I L A Q

P

V E

V E

T R

R E A

A N I D S K K L L L I I A V V

The skilled artisan will realize that these and other amino acidsubstitutions in the DDD or AD amino acid sequences may be utilized toproduce alternative species within the genus of AD or DDD moieties,using techniques that are standard in the field and only routineexperimentation.

Antibody Allotypes

Immunogenicity of therapeutic antibodies is associated with increasedrisk of infusion reactions and decreased duration of therapeuticresponse (Baert et al., 2003, N Engl J Med 348:602-08). The extent towhich therapeutic antibodies induce an immune response in the host maybe determined in part by the allotype of the antibody (Stickler et al.,2011, Genes and Immunity 12:213-21). Antibody allotype is related toamino acid sequence variations at specific locations in the constantregion sequences of the antibody. The allotypes of IgG antibodiescontaining a heavy chain γ-type constant region are designated as Gmallotypes (1976, J Immunol 117:1056-59).

For the common IgG1 human antibodies, the most prevalent allotype isG1m1 (Stickler et al., 2011, Genes and Immunity 12:213-21). However, theG1m3 allotype also occurs frequently in Caucasians (Id.). It has beenreported that G1m1 antibodies contain allotypic sequences that tend toinduce an immune response when administered to non-G1m1 (nG1m1)recipients, such as G1m3 patients (Id.). Non-G1m1 allotype antibodiesare not as immunogenic when administered to G1m1 patients (Id.).

The human G1m1 allotype comprises the amino acids aspartic acid at Kabatposition 356 and leucine at Kabat position 358 in the CH3 sequence ofthe heavy chain IgG1. The nG1m1 allotype comprises the amino acidsglutamic acid at Kabat position 356 and methionine at Kabat position358. Both G1m1 and nG1m1 allotypes comprise a glutamic acid residue atKabat position 357 and the allotypes are sometimes referred to as DELand EEM allotypes. A non-limiting example of the heavy chain constantregion sequences for G1m1 and nG1m1 allotype antibodies is shown for theexemplary antibodies rituximab (SEQ ID NO:19) and veltuzumab (SEQ IDNO:20).

Rituximab heavy chain variable region sequence (SEQ ID NO: 19)ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Veltuzumab heavy chain variable region(SEQ ID NO: 20) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Jefferis and Lefranc (2009, mAbs 1:1-7) reviewed sequence variationscharacteristic of IgG allotypes and their effect on immunogenicity. Theyreported that the G1m3 allotype is characterized by an arginine residueat Kabat position 214, compared to a lysine residue at Kabat 214 in theG1m17 allotype. The nG1m1,2 allotype was characterized by glutamic acidat Kabat position 356, methionine at Kabat position 358 and alanine atKabat position 431. The G1m1,2 allotype was characterized by asparticacid at Kabat position 356, leucine at Kabat position 358 and glycine atKabat position 431. In addition to heavy chain constant region sequencevariants, Jefferis and Lefranc (2009) reported allotypic variants in thekappa light chain constant region, with the Km1 allotype characterizedby valine at Kabat position 153 and leucine at Kabat position 191, theKm1,2 allotype by alanine at Kabat position 153 and leucine at Kabatposition 191, and the Km3 allotypoe characterized by alanine at Kabatposition 153 and valine at Kabat position 191.

With regard to therapeutic antibodies, veltuzumab and rituximab are,respectively, humanized and chimeric IgG1 antibodies against CD20, ofuse for therapy of a wide variety of hematological malignancies. Table 5compares the allotype sequences of rituximab vs. veltuzumab. As shown inTable 5, rituximab (G1m17,1) is a DEL allotype IgG1, with an additionalsequence variation at Kabat position 214 (heavy chain CH1) of lysine inrituximab vs. arginine in veltuzumab. It has been reported thatveltuzumab is less immunogenic in subjects than rituximab (see, e.g.,Morchhauser et al., 2009, J Clin Oncol 27:3346-53; Goldenberg et al.,2009, Blood 113:1062-70; Robak & Robak, 2011, BioDrugs 25:13-25), aneffect that has been attributed to the difference between humanized andchimeric antibodies. However, the difference in allotypes between theEEM and DEL allotypes likely also accounts for the lower immunogenicityof veltuzumab.

TABLE 5 Allotypes of Rituximab vs. Veltuzumab Heavy chain position andassociated allotypes Complete allotype 214 (allotype) 356/358 (allotype)431 (allotype) Rituximab G1m17,1 K 17 D/L 1 A — Veltuzumab G1m3 R 3 E/M— A —

In order to reduce the immunogenicity of therapeutic antibodies inindividuals of nG1m1 genotype, it is desirable to select the allotype ofthe antibody to correspond to the G1m3 allotype, characterized byarginine at Kabat 214, and the nG1m1,2 null-allotype, characterized byglutamic acid at Kabat position 356, methionine at Kabat position 358and alanine at Kabat position 431. Surprisingly, it was found thatrepeated subcutaneous administration of G1m3 antibodies over a longperiod of time did not result in a significant immune response. Inalternative embodiments, the human IgG4 heavy chain in common with theG1m3 allotype has arginine at Kabat 214, glutamic acid at Kabat 356,methionine at Kabat 359 and alanine at Kabat 431. Since immunogenicityappears to relate at least in part to the residues at those locations,use of the human IgG4 heavy chain constant region sequence fortherapeutic antibodies is also a preferred embodiment. Combinations ofG1m3 IgG1 antibodies with IgG4 antibodies may also be of use fortherapeutic administration.

Amino Acid Substitutions

In alternative embodiments, the disclosed methods and compositions mayinvolve production and use of proteins or peptides with one or moresubstituted amino acid residues. The skilled artisan will be aware that,in general, amino acid substitutions typically involve the replacementof an amino acid with another amino acid of relatively similarproperties (i.e., conservative amino acid substitutions). The propertiesof the various amino acids and effect of amino acid substitution onprotein structure and function have been the subject of extensive studyand knowledge in the art.

For example, the hydropathic index of amino acids may be considered(Kyte & Doolittle, 1982, J. Mol. Biol., 157:105-132). The relativehydropathic character of the amino acid contributes to the secondarystructure of the resultant protein, which in turn defines theinteraction of the protein with other molecules. Each amino acid hasbeen assigned a hydropathic index on the basis of its hydrophobicity andcharge characteristics (Kyte & Doolittle, 1982), these are: isoleucine(+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8);cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine(−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine(−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine(−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9); and arginine(−4.5). In making conservative substitutions, the use of amino acidswhose hydropathic indices are within ±2 is preferred, within ±1 are morepreferred, and within ±0.5 are even more preferred.

Amino acid substitution may also take into account the hydrophilicity ofthe amino acid residue (e.g., U.S. Pat. No. 4,554,101). Hydrophilicityvalues have been assigned to amino acid residues: arginine (+3.0);lysine (+3.0); aspartate (+3.0); glutamate (+3.0); serine (+0.3);asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4);proline (−0.5+−0.1); alanine (−0.5); histidine (−0.5); cysteine (−1.0);methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8);tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4). Replacement ofamino acids with others of similar hydrophilicity is preferred.

Other considerations include the size of the amino acid side chain. Forexample, it would generally not be preferred to replace an amino acidwith a compact side chain, such as glycine or serine, with an amino acidwith a bulky side chain, e.g., tryptophan or tyrosine. The effect ofvarious amino acid residues on protein secondary structure is also aconsideration. Through empirical study, the effect of different aminoacid residues on the tendency of protein domains to adopt analpha-helical, beta-sheet or reverse turn secondary structure has beendetermined and is known in the art (see, e.g., Chou & Fasman, 1974,Biochemistry, 13:222-245; 1978, Ann. Rev. Biochem., 47: 251-276; 1979,Biophys. J., 26:367-384).

Based on such considerations and extensive empirical study, tables ofconservative amino acid substitutions have been constructed and areknown in the art. For example: arginine and lysine; glutamate andaspartate; serine and threonine; glutamine and asparagine; and valine,leucine and isoleucine. Alternatively: Ala (A) leu, ile, val; Arg (R)gln, asn, lys; Asn (N) his, asp, lys, arg, gln; Asp (D) asn, glu; Cys(C) ala, ser; Gln (Q) glu, asn; Glu (E) gln, asp; Gly (G) ala; His (H)asn, gln, lys, arg; Ile (I) val, met, ala, phe, leu; Leu (L) val, met,ala, phe, ile; Lys (K) gln, asn, arg; Met (M) phe, ile, leu; Phe (F)leu, val, ile, ala, tyr; Pro (P) ala; Ser (S), thr; Thr (T) ser; Trp (W)phe, tyr; Tyr (Y) trp, phe, thr, ser; Val (V) ile, leu, met, phe, ala.

Other considerations for amino acid substitutions include whether or notthe residue is located in the interior of a protein or is solventexposed. For interior residues, conservative substitutions wouldinclude: Asp and Asn; Ser and Thr; Ser and Ala; Thr and Ala; Ala andGly; Ile and Val; Val and Leu; Leu and Ile; Leu and Met; Phe and Tyr;Tyr and Trp. (See, e.g., PROWL website at rockefeller.edu) For solventexposed residues, conservative substitutions would include: Asp and Asn;Asp and Glu; Glu and Gln; Glu and Ala; Gly and Asn; Ala and Pro; Ala andGly; Ala and Ser; Ala and Lys; Ser and Thr; Lys and Arg; Val and Leu;Leu and Ile; Ile and Val; Phe and Tyr. (Id.) Various matrices have beenconstructed to assist in selection of amino acid substitutions, such asthe PAM250 scoring matrix, Dayhoff matrix, Grantham matrix, McLachlanmatrix, Doolittle matrix, Henikoff matrix, Miyata matrix, Fitch matrix,Jones matrix, Rao matrix, Levin matrix and Risler matrix (Idem.)

In determining amino acid substitutions, one may also consider theexistence of intermolecular or intramolecular bonds, such as formationof ionic bonds (salt bridges) between positively charged residues (e.g.,His, Arg, Lys) and negatively charged residues (e.g., Asp, Glu) ordisulfide bonds between nearby cysteine residues.

Methods of substituting any amino acid for any other amino acid in anencoded protein sequence are well known and a matter of routineexperimentation for the skilled artisan, for example by the technique ofsite-directed mutagenesis or by synthesis and assembly ofoligonucleotides encoding an amino acid substitution and splicing intoan expression vector construct.

Avimers

In certain embodiments, the binding moieties described herein maycomprise one or more avimer sequences. Avimers are a class of bindingproteins somewhat similar to antibodies in their affinities andspecificities for various target molecules. They were developed fromhuman extracellular receptor domains by in vitro exon shuffling andphage display. (Silverman et al., 2005, Nat. Biotechnol. 23:1493-94;Silverman et al., 2006, Nat. Biotechnol. 24:220). The resultingmultidomain proteins may comprise multiple independent binding domains,that may exhibit improved affinity (in some cases sub-nanomolar) andspecificity compared with single-epitope binding proteins. (Id.) Invarious embodiments, avimers may be attached to, for example, DDD and/orAD sequences for use in the claimed methods and compositions. Additionaldetails concerning methods of construction and use of avimers aredisclosed, for example, in U.S. Patent Application Publication Nos.20040175756, 20050048512, 20050053973, 20050089932 and 20050221384, theExamples section of each of which is incorporated herein by reference.

Phage Display

Certain embodiments of the claimed compositions and/or methods mayconcern binding peptides and/or peptide mimetics of various targetmolecules, cells or tissues. Binding peptides may be identified by anymethod known in the art, including but not limiting to the phage displaytechnique. Various methods of phage display and techniques for producingdiverse populations of peptides are well known in the art. For example,U.S. Pat. Nos. 5,223,409; 5,622,699 and 6,068,829 disclose methods forpreparing a phage library. The phage display technique involvesgenetically manipulating bacteriophage so that small peptides can beexpressed on their surface (Smith and Scott, 1985, Science228:1315-1317; Smith and Scott, 1993, Meth. Enzymol. 21:228-257). Inaddition to peptides, larger protein domains such as single-chainantibodies may also be displayed on the surface of phage particles (Arapet al., 1998, Science 279:377-380).

Targeting amino acid sequences selective for a given organ, tissue, celltype or target molecule may be isolated by panning (Pasqualini andRuoslahti, 1996, Nature 380:364-366; Pasqualini, 1999, The Quart. J.Nucl. Med. 43:159-162). In brief, a library of phage containing putativetargeting peptides is administered to an intact organism or to isolatedorgans, tissues, cell types or target molecules and samples containingbound phage are collected. Phage that bind to a target may be elutedfrom a target organ, tissue, cell type or target molecule and thenamplified by growing them in host bacteria.

In certain embodiments, the phage may be propagated in host bacteriabetween rounds of panning. Rather than being lysed by the phage, thebacteria may instead secrete multiple copies of phage that display aparticular insert. If desired, the amplified phage may be exposed to thetarget organs, tissues, cell types or target molecule again andcollected for additional rounds of panning. Multiple rounds of panningmay be performed until a population of selective or specific binders isobtained. The amino acid sequence of the peptides may be determined bysequencing the DNA corresponding to the targeting peptide insert in thephage genome. The identified targeting peptide may then be produced as asynthetic peptide by standard protein chemistry techniques (Arap et al.,1998, Smith et al., 1985).

In some embodiments, a subtraction protocol may be used to furtherreduce background phage binding. The purpose of subtraction is to removephage from the library that bind to targets other than the target ofinterest. In alternative embodiments, the phage library may beprescreened against a control cell, tissue or organ. For example,tumor-binding peptides may be identified after prescreening a libraryagainst a control normal cell line. After subtraction the library may bescreened against the molecule, cell, tissue or organ of interest. Othermethods of subtraction protocols are known and may be used in thepractice of the claimed methods, for example as disclosed in U.S. Pat.Nos. 5,840,841, 5,705,610, 5,670,312 and 5,492,807.

Aptamers

In certain embodiments, a targeting moiety of use may be an aptamer.Methods of constructing and determining the binding characteristics ofaptamers are well known in the art. For example, such techniques aredescribed in U.S. Pat. Nos. 5,582,981, 5,595,877 and 5,637,459, theExamples section of each incorporated herein by reference. Methods forpreparation and screening of aptamers that bind to particular targets ofinterest are well known, for example U.S. Pat. Nos. 5,475,096 and5,270,163, the Examples section of each incorporated herein byreference.

Aptamers may be prepared by any known method, including synthetic,recombinant, and purification methods, and may be used alone or incombination with other ligands specific for the same target. In general,a minimum of approximately 3 nucleotides, preferably at least 5nucleotides, are necessary to effect specific binding. Aptamers ofsequences shorter than 10 bases may be feasible, although aptamers of10, 20, 30 or 40 nucleotides may be preferred.

Aptamers may be isolated, sequenced, and/or amplified or synthesized asconventional DNA or RNA molecules. Alternatively, aptamers of interestmay comprise modified oligomers. Any of the hydroxyl groups ordinarilypresent in aptamers may be replaced by phosphonate groups, phosphategroups, protected by a standard protecting group, or activated toprepare additional linkages to other nucleotides, or may be conjugatedto solid supports. One or more phosphodiester linkages may be replacedby alternative linking groups, such as P(O)O replaced by P(O)S, P(O)NR₂,P(O)R, P(O)OR′, CO, or CNR₂, wherein R is H or alkyl (1-20C) and R′ isalkyl (1-20C); in addition, this group may be attached to adjacentnucleotides through O or S. Not all linkages in an oligomer need to beidentical.

Affibodies and Fynomers

Certain alternative embodiments may utilize affibodies in place ofantibodies. Affibodies are commercially available from Affibody AB(Solna, Sweden). Affibodies are small proteins that function as antibodymimetics and are of use in binding target molecules. Affibodies weredeveloped by combinatorial engineering on an alpha helical proteinscaffold (Nord et al., 1995, Protein Eng 8:601-8; Nord et al., 1997, NatBiotechnol 15:772-77). The affibody design is based on a three helixbundle structure comprising the IgG binding domain of protein A (Nord etal., 1995; 1997). Affibodies with a wide range of binding affinities maybe produced by randomization of thirteen amino acids involved in the Fcbinding activity of the bacterial protein A (Nord et al., 1995; 1997).After randomization, the PCR amplified library was cloned into aphagemid vector for screening by phage display of the mutant proteins.The phage display library may be screened against any known antigen,using standard phage display screening techniques (e.g., Pasqualini andRuoslahti, 1996, Nature 380:364-366; Pasqualini, 1999, Quart. J. Nucl.Med. 43:159-162), in order to identify one or more affibodies againstthe target antigen.

A ¹⁷⁷Lu-labeled affibody specific for HER2/neu has been demonstrated totarget HER2-expressing xenografts in vivo (Tolmachev et al., 2007,Cancer Res 67:2773-82). Although renal toxicity due to accumulation ofthe low molecular weight radiolabeled compound was initially a problem,reversible binding to albumin reduced renal accumulation, enablingradionuclide-based therapy with labeled affibody (Id.).

The feasibility of using radiolabeled affibodies for in vivo tumorimaging has been recently demonstrated (Tolmachev et al., 2011,Bioconjugate Chem 22:894-902). A maleimide-derivatized NOTA wasconjugated to the anti-HER2 affibody and radiolabeled with ¹¹¹In (Id.).Administration to mice bearing the HER2-expressing DU-145 xenograft,followed by gamma camera imaging, allowed visualization of the xenograft(Id.).

Fynomers can also bind to target antigens with a similar affinity andspecificity to antibodies. Fynomers are based on the human Fyn SH3domain as a scaffold for assembly of binding molecules. The Fyn SH3domain is a fully human, 63 amino acid protein that can be produced inbacteria with high yields. Fynomers may be linked together to yield amultispecific binding protein with affinities for two or more differentantigen targets. Fynomers are commercially available from COVAGEN AG(Zurich, Switzerland).

The skilled artisan will realize that affibodies or fynomers may be usedas targeting molecules in the practice of the claimed methods andcompositions.

Immunoconjugates

In certain embodiments, a cytotoxic drug or other therapeutic ordiagnostic agent may be covalently attached to an antibody or antibodyfragment to form an immunoconjugate. In some embodiments, a drug orother agent may be attached to an antibody or fragment thereof via acarrier moiety. Carrier moieties may be attached, for example to reducedSH groups and/or to carbohydrate side chains. A carrier moiety can beattached at the hinge region of a reduced antibody component viadisulfide bond formation. Alternatively, such agents can be attachedusing a heterobifunctional cross-linker, such as N-succinyl3-(2-pyridyldithio)propionate (SPDP). Yu et al., Int. J. Cancer 56: 244(1994). General techniques for such conjugation are well-known in theart. See, for example, Wong, CHEMISTRY OF PROTEIN CONJUGATION ANDCROSS-LINKING (CRC Press 1991); Upeslacis et al., “Modification ofAntibodies by Chemical Methods,” in MONOCLONAL ANTIBODIES: PRINCIPLESAND APPLICATIONS, Birch et al. (eds.), pages 187-230 (Wiley-Liss, Inc.1995); Price, “Production and Characterization of SyntheticPeptide-Derived Antibodies,” in MONOCLONAL ANTIBODIES: PRODUCTION,ENGINEERING AND CLINICAL APPLICATION, Ritter et al. (eds.), pages 60-84(Cambridge University Press 1995). Alternatively, the carrier moiety canbe conjugated via a carbohydrate moiety in the Fc region of theantibody.

Methods for conjugating functional groups to antibodies via an antibodycarbohydrate moiety are well-known to those of skill in the art. See,for example, Shih et al., Int. J. Cancer 41: 832 (1988); Shih et al.,Int. J. Cancer 46: 1101 (1990); and Shih et al., U.S. Pat. No.5,057,313, the Examples section of which is incorporated herein byreference. The general method involves reacting an antibody having anoxidized carbohydrate portion with a carrier polymer that has at leastone free amine function. This reaction results in an initial Schiff base(imine) linkage, which can be stabilized by reduction to a secondaryamine to form the final conjugate.

The Fc region may be absent if the antibody component of the ADC is anantibody fragment. However, it is possible to introduce a carbohydratemoiety into the light chain variable region of a full length antibody orantibody fragment. See, for example, Leung et al., J. Immunol. 154: 5919(1995); U.S. Pat. Nos. 5,443,953 and 6,254,868, the Examples section ofwhich is incorporated herein by reference. The engineered carbohydratemoiety is used to attach the therapeutic or diagnostic agent.

An alternative method for attaching carrier moieties to a targetingmolecule involves use of click chemistry reactions. The click chemistryapproach was originally conceived as a method to rapidly generatecomplex substances by joining small subunits together in a modularfashion. (See, e.g., Kolb et al., 2004, Angew Chem Int Ed 40:3004-31;Evans, 2007, Aust J Chem 60:384-95.) Various forms of click chemistryreaction are known in the art, such as the Huisgen 1,3-dipolarcycloaddition copper catalyzed reaction (Tornoe et al., 2002, J OrganicChem 67:3057-64), which is often referred to as the “click reaction.”Other alternatives include cycloaddition reactions such as theDiels-Alder, nucleophilic substitution reactions (especially to smallstrained rings like epoxy and aziridine compounds), carbonyl chemistryformation of urea compounds and reactions involving carbon-carbon doublebonds, such as alkynes in thiol-yne reactions.

The azide alkyne Huisgen cycloaddition reaction uses a copper catalystin the presence of a reducing agent to catalyze the reaction of aterminal alkyne group attached to a first molecule. In the presence of asecond molecule comprising an azide moiety, the azide reacts with theactivated alkyne to form a 1,4-disubstituted 1,2,3-triazole. The coppercatalyzed reaction occurs at room temperature and is sufficientlyspecific that purification of the reaction product is often notrequired. (Rostovstev et al., 2002, Angew Chem Int Ed 41:2596; Tornoe etal., 2002, J Org Chem 67:3057.) The azide and alkyne functional groupsare largely inert towards biomolecules in aqueous medium, allowing thereaction to occur in complex solutions. The triazole formed ischemically stable and is not subject to enzymatic cleavage, making theclick chemistry product highly stable in biological systems. Althoughthe copper catalyst is toxic to living cells, the copper-based clickchemistry reaction may be used in vitro for immunoconjugate formation.

A copper-free click reaction has been proposed for covalent modificationof biomolecules. (See, e.g., Agard et al., 2004, J Am Chem Soc126:15046-47.) The copper-free reaction uses ring strain in place of thecopper catalyst to promote a [3+2] azide-alkyne cycloaddition reaction(Id.) For example, cyclooctyne is an 8-carbon ring structure comprisingan internal alkyne bond. The closed ring structure induces a substantialbond angle deformation of the acetylene, which is highly reactive withazide groups to form a triazole. Thus, cyclooctyne derivatives may beused for copper-free click reactions (Id.)

Another type of copper-free click reaction was reported by Ning et al.(2010, Angew Chem Int Ed 49:3065-68), involving strain-promotedalkyne-nitrone cycloaddition. To address the slow rate of the originalcyclooctyne reaction, electron-withdrawing groups are attached adjacentto the triple bond (Id.) Examples of such substituted cyclooctynesinclude difluorinated cyclooctynes, 4-dibenzocyclooctynol andazacyclooctyne (Id.) An alternative copper-free reaction involvedstrain-promoted alkyne-nitrone cycloaddition to give N-alkylatedisoxazolines (Id.) The reaction was reported to have exceptionally fastreaction kinetics and was used in a one-pot three-step protocol forsite-specific modification of peptides and proteins (Id.) Nitrones wereprepared by the condensation of appropriate aldehydes withN-methylhydroxylamine and the cycloaddition reaction took place in amixture of acetonitrile and water (Id.) These and other known clickchemistry reactions may be used to attach carrier moieties to antibodiesin vitro.

Agard et al. (2004, J Am Chem Soc 126:15046-47) demonstrated that arecombinant glycoprotein expressed in CHO cells in the presence ofperacetylated N-azidoacetylmannosamine resulted in the bioincorporationof the corresponding N-azidoacetyl sialic acid in the carbohydrates ofthe glycoprotein. The azido-derivatized glycoprotein reactedspecifically with a biotinylated cyclooctyne to form a biotinylatedglycoprotein, while control glycoprotein without the azido moietyremained unlabeled (Id.) Laughlin et al. (2008, Science 320:664-667)used a similar technique to metabolically label cell-surface glycans inzebrafish embryos incubated with peracetylatedN-azidoacetylgalactosamine. The azido-derivatized glycans reacted withdifluorinated cyclooctyne (DIFO) reagents to allow visualization ofglycans in vivo.

The Diels-Alder reaction has also been used for in vivo labeling ofmolecules. Rossin et al. (2010, Angew Chem Int Ed 49:3375-78) reported a52% yield in vivo between a tumor-localized anti-TAG72 (CC49) antibodycarrying a trans-cyclooctene (TCO) reactive moiety and an ¹¹¹In-labeledtetrazine DOTA derivative. The TCO-labeled CC49 antibody wasadministered to mice bearing colon cancer xenografts, followed 1 daylater by injection of ¹¹¹In-labeled tetrazine probe (Id.) The reactionof radiolabeled probe with tumor localized antibody resulted inpronounced radioactivity localization in the tumor, as demonstrated bySPECT imaging of live mice three hours after injection of radiolabeledprobe, with a tumor-to-muscle ratio of 13:1 (Id.) The results confirmedthe in vivo chemical reaction of the TCO and tetrazine-labeledmolecules.

Antibody labeling techniques using biological incorporation of labelingmoieties are further disclosed in U.S. Pat. No. 6,953,675 (the Examplessection of which is incorporated herein by reference). Such “landscaped”antibodies were prepared to have reactive ketone groups on glycosylatedsites. The method involved expressing cells transfected with anexpression vector encoding an antibody with one or more N-glycosylationsites in the CH1 or Vκ domain in culture medium comprising a ketonederivative of a saccharide or saccharide precursor. Ketone-derivatizedsaccharides or precursors included N-levulinoyl mannosamine andN-levulinoyl fucose. The landscaped antibodies were subsequently reactedwith agents comprising a ketone-reactive moiety, such as hydrazide,hydrazine, hydroxylamino or thiosemicarbazide groups, to form a labeledtargeting molecule. Exemplary agents attached to the landscapedantibodies included chelating agents like DTPA, large drug moleculessuch as doxorubicin-dextran, and acyl-hydrazide containing peptides. Thelandscaping technique is not limited to producing antibodies comprisingketone moieties, but may be used instead to introduce a click chemistryreactive group, such as a nitrone, an azide or a cyclooctyne, onto anantibody or other biological molecule.

Modifications of click chemistry reactions are suitable for use in vitroor in vivo. Reactive targeting molecule may be formed either by eitherchemical conjugation or by biological incorporation. The targetingmolecule, such as an antibody or antibody fragment, may be activatedwith an azido moiety, a substituted cyclooctyne or alkyne group, or anitrone moiety. Where the targeting molecule comprises an azido ornitrone group, the corresponding targetable construct will comprise asubstituted cyclooctyne or alkyne group, and vice versa. Such activatedmolecules may be made by metabolic incorporation in living cells, asdiscussed above.

Alternatively, methods of chemical conjugation of such moieties tobiomolecules are well known in the art, and any such known method may beutilized. General methods of immunoconjugate formation are disclosed,for example, in U.S. Pat. Nos. 4,699,784; 4,824,659; 5,525,338;5,677,427; 5,697,902; 5,716,595; 6,071,490; 6,187,284; 6,306,393;6,548,275; 6,653,104; 6,962,702; 7,033,572; 7,147,856; and 7,259,240,the Examples section of each incorporated herein by reference.

The preferred conjugation protocol is based on a thiol-maleimide, athiol-vinylsulfone, a thiol-bromoacetamide, or a thiol-iodoacetamidereaction that is facile at neutral or acidic pH. This obviates the needfor higher pH conditions for conjugations as, for instance, would benecessitated when using active esters. Further details of exemplaryconjugation protocols are described below in the Examples section.

Therapeutic Treatment

In another aspect, the invention relates to a method of treating asubject, comprising administering to a subject a therapeuticallyeffective amount of an antibody-drug conjugate (ADC) as describedherein. Diseases that may be treated with the ADCs described hereininclude, but are not limited to B-cell malignancies (e.g., non-Hodgkin'slymphoma, mantle cell lymphoma, multiple myeloma, Hodgkin's lymphoma,diffuse large B cell lymphoma, Burkitt lymphoma, follicular lymphoma,acute lymphocytic leukemia, chronic lymphocytic leukemia, hairy cellleukemia) using, for example an anti-CD22 antibody such as the hLL2 MAb(epratuzumab, see U.S. Pat. No. 6,183,744), against another CD22 epitope(hRFB4) or antibodies against other B cell antigens, such as CD19, CD20,CD21, CD22, CD23, CD37, CD40, CD40L, CD52, CD74, CD80 or HLA-DR. Otherdiseases include, but are not limited to, adenocarcinomas ofendodermally-derived digestive system epithelia, cancers such as breastcancer and non-small cell lung cancer, and other carcinomas, sarcomas,glial tumors, myeloid leukemias, etc. In particular, antibodies againstan antigen, e.g., an oncofetal antigen, produced by or associated with amalignant solid tumor or hematopoietic neoplasm, e.g., agastrointestinal, stomach, colon, esophageal, liver, lung, breast,pancreatic, liver, prostate, ovarian, testicular, brain, bone orlymphatic tumor, a sarcoma or a melanoma, are advantageously used. Suchtherapeutics can be given once or repeatedly, depending on the diseasestate and tolerability of the conjugate, and can also be used optionallyin combination with other therapeutic modalities, such as surgery,external radiation, radioimmunotherapy, immunotherapy, chemotherapy,antisense therapy, interference RNA therapy, gene therapy, and the like.Each combination will be adapted to the tumor type, stage, patientcondition and prior therapy, and other factors considered by themanaging physician.

As used herein, the term “subject” refers to any animal (i.e.,vertebrates and invertebrates) including, but not limited to mammals,including humans. It is not intended that the term be limited to aparticular age or sex. Thus, adult and newborn subjects, as well asfetuses, whether male or female, are encompassed by the term. Dosesgiven herein are for humans, but can be adjusted to the size of othermammals, as well as children, in accordance with weight or square metersize.

In a preferred embodiment, therapeutic conjugates comprising ananti-TROP-2 antibody such as the hRS7 MAb can be used to treatcarcinomas such as carcinomas of the esophagus, pancreas, lung, stomach,colon and rectum, urinary bladder, breast, ovary, uterus, kidney andprostate, as disclosed in U.S. Pat. Nos. 7,238,785; 7,517,964 and8,084,583, the Examples section of which is incorporated herein byreference. An hRS7 antibody is a humanized antibody that comprises lightchain complementarity-determining region (CDR) sequences CDR1(KASQDVSIAVA, SEQ ID NO:21); CDR2 (SASYRYT, SEQ ID NO:22); and CDR3(QQHYITPLT, SEQ ID NO:23) and heavy chain CDR sequences CDR1 (NYGMN, SEQID NO:24); CDR2 (WINTYTGEPTYTDDFKG, SEQ ID NO:25) and CDR3(GGFGSSYWYFDV, SEQ ID NO:26)

In another preferred embodiment, therapeutic conjugates comprising ananti-CEACAM5 antibody (e.g., hMN-14, labretuzumab) and/or ananti-CEACAM6 antibody may be used to treat any of a variety of cancersthat express CEACAM5 and/or CEACAM6, as disclosed in U.S. Pat. Nos.7,541,440; 7,951,369; 5,874,540; 6,676,924 and 8,267,865, the Examplessection of each incorporated herein by reference. Solid tumors that maybe treated using anti-CEACAM5, anti-CEACAM6, or a combination of the twoinclude but are not limited to breast, lung, pancreatic, esophageal,medullary thyroid, ovarian, colon, rectum, urinary bladder, mouth andstomach cancers. A majority of carcinomas, including gastrointestinal,respiratory, genitourinary and breast cancers express CEACAM5 and may betreated with the subject immunoconjugates. An hMN-14 antibody is ahumanized antibody that comprises light chain variable region CDRsequences CDR1 (KASQDVGTSVA; SEQ ID NO:27), CDR2 (WTSTRHT; SEQ IDNO:28), and CDR3 (QQYSLYRS; SEQ ID NO:29), and the heavy chain variableregion CDR sequences CDR1 (TYWMS; SEQ ID NO:30), CDR2(EIHPDSSTINYAPSLKD; SEQ ID NO:31) and CDR3 (LYFGFPWFAY; SEQ ID NO:32).

In another preferred embodiment, therapeutic conjugates comprising ananti-CD74 antibody (e.g., hLL1, milatuzumab, disclosed in U.S. Pat. Nos.7,074,403; 7,312,318; 7,772,373; 7,919,087 and 7,931,903, the Examplessection of each incorporated herein by reference) may be used to treatany of a variety of cancers that express CD74, including but not limitedto renal, lung, intestinal, stomach, breast, prostate or ovarian cancer,as well as several hematological cancers, such as multiple myeloma,chronic lymphocytic leukemia, acute lymphoblastic leukemia, non-Hodgkinlymphoma, and Hodgkin lymphoma. An hLL1 antibody is a humanized antibodycomprising the light chain CDR sequences CDR1 (RSSQSLVHRNGNTYLH; SEQ IDNO:33), CDR2 (TVSNRFS; SEQ ID NO:34), and CDR3 (SQSSHVPPT; SEQ ID NO:35)and the heavy chain variable region CDR sequences CDR1 (NYGVN; SEQ IDNO:36), CDR2 (WINPNTGEPTFDDDFKG; SEQ ID NO:37), and CDR3 (SRGKNEAWFAY;SEQ ID NO:38).

In another preferred embodiment, therapeutic conjugates comprising ananti-CD22 antibody (e.g., hLL2, epratuzumab, disclosed in U.S. Pat. Nos.5,789,554; 6,183,744; 6,187,287; 6,306,393; 7,074,403 and 7,641,901, theExamples section of each incorporated herein by reference, or thechimeric or humanized RFB4 antibody) may be used to treat any of avariety of cancers that express CD22, including but not limited toindolent forms of B-cell lymphomas, aggressive forms of B-celllymphomas, chronic lymphatic leukemias, acute lymphatic leukemias,non-Hodgkin's lymphoma, Hodgkin's lymphoma, Burkitt lymphoma, follicularlymphoma or diffuse B-cell lymphoma. An hLL2 antibody is a humanizedantibody comprising light chain CDR sequences CDR1 (KSSQSVLYSANHKYLA,SEQ ID NO:39), CDR2 (WASTRES, SEQ ID NO:40), and CDR3 (HQYLSSWTF, SEQ IDNO:41) and the heavy chain CDR sequences CDR1 (SYWLH, SEQ ID NO:42),CDR2 (YINPRNDYTEYNQNFKD, SEQ ID NO:43), and CDR3 (RDITTFY, SEQ ID NO:44)

In another preferred embodiment, therapeutic conjugates comprising ananti-HLA-DR MAb, such as hL243, can be used to treat lymphoma, leukemia,cancers of the skin, esophagus, stomach, colon, rectum, pancreas, lung,breast, ovary, bladder, endometrium, cervix, testes, kidney, liver,melanoma or other HLA-DR-producing tumors, as disclosed in U.S. Pat. No.7,612,180, the Examples section of which is incorporated herein byreference. An hL243 antibody is a humanized antibody comprising theheavy chain CDR sequences CDR1 (NYGMN, SEQ ID NO:45), CDR2(WINTYTREPTYADDFKG, SEQ ID NO:46), and CDR3 (DITAVVPTGFDY, SEQ ID NO:47)and light chain CDR sequences CDR1 (RASENIYSNLA, SEQ ID NO:48), CDR2(AASNLAD, SEQ ID NO:49), and CDR3 (QHFWTTPWA, SEQ ID NO:50).

In another preferred embodiment, therapeutic conjugates comprising ananti-CD20 MAb, such as veltuzumab (hA20), 1F5, obinutuzumab (GA101), orrituximab, can be used to treat lymphoma, leukemia, immunethrombocytopenic purpura, systemic lupus erythematosus, Sjögren'ssyndrome, Evans syndrome, arthritis, arteritis, pemphigus vulgaris,renal graft rejection, cardiac graft rejection, rheumatoid arthritis,Burkitt lymphoma, non-Hodgkin's lymphoma, follicular lymphoma, smalllymphocytic lymphoma, diffuse B-cell lymphoma, marginal zone lymphoma,chronic lymphocytic leukemia, acute lymphocytic leukemia, Type Idiabetes mellitus, GVHD, multiple sclerosis or multiple myeloma, asdisclosed in U.S. Pat. Nos. 7,435,803 or 8,287,864, the Examples sectionof each incorporated herein by reference. An hA20 (veltuzumab) antibodyis a humanized antibody comprising the light chain CDR sequences CDRL1(RASSSVSYIH, SEQ ID NO:51), CDRL2 (ATSNLAS, SEQ ID NO:52) and CDRL3(QQWTSNPPT, SEQ ID NO:53) and heavy chain CDR sequences CDRH1 (SYNMH,SEQ ID NO:54), CDRH2 (AIYPGNGDTSYNQKFKG, SEQ ID NO:55) and CDRH3(STYYGGDWYFDV, SEQ ID NO:56).

In another preferred embodiment, therapeutic conjugates comprisinganti-tenascin antibodies can be used to treat hematopoietic and solidtumors, and conjugates comprising antibodies to tenascin can be used totreat solid tumors, preferably brain cancers like glioblastomas.

In a preferred embodiment, the antibodies that are used in the treatmentof human disease are human or humanized (CDR-grafted) versions ofantibodies; although murine and chimeric versions of antibodies can beused. Same species IgG molecules as delivery agents are mostly preferredto minimize immune responses. This is particularly important whenconsidering repeat treatments. For humans, a human or humanized IgGantibody is less likely to generate an anti-IgG immune response frompatients. Antibodies such as hLL1 and hLL2 rapidly internalize afterbinding to internalizing antigen on target cells, which means that thechemotherapeutic drug being carried is rapidly internalized into cellsas well. However, antibodies that have slower rates of internalizationcan also be used to effect selective therapy.

In a preferred embodiment, a more effective incorporation into cells canbe accomplished by using multivalent, multispecific or multivalent,monospecific antibodies. Examples of such bivalent and bispecificantibodies are found in U.S. Pat. Nos. 7,387,772; 7,300,655; 7,238,785;and 7,282,567, the Examples section of each of which is incorporatedherein by reference. These multivalent or multispecific antibodies areparticularly preferred in the targeting of cancers and infectiousorganisms (pathogens), which express multiple antigen targets and evenmultiple epitopes of the same antigen target, but which often evadeantibody targeting and sufficient binding for immunotherapy because ofinsufficient expression or availability of a single antigen target onthe cell or pathogen. By targeting multiple antigens or epitopes, saidantibodies show a higher binding and residence time on the target, thusaffording a higher saturation with the drug being targeted in thisinvention.

In another preferred embodiment, the therapeutic conjugates can be usedto treat autoimmune disease or immune system dysfunction (e.g.,graft-versus-host disease, organ transplant rejection). Antibodies ofuse to treat autoimmune/immune dysfunction disease may bind to exemplaryantigens including, but not limited to, BCL-1, BCL-2, BCL-6, CD1a, CD2,CD3, CD4, CD5, CD7, CD8, CD10, CD11b, CD11c, CD13, CD14, CD15, CD16,CD19, CD20, CD21, CD22, CD23, CD25, CD33, CD34, CD38, CD40, CD40L,CD41a, CD43, CD45, CD55, CD56, CCD57, CD59, CD64, CD71, CD74, CD79a,CD79b, CD117, CD138, FMC-7 and HLA-DR. Antibodies that bind to these andother target antigens, discussed above, may be used to treat autoimmuneor immune dysfunction diseases. Autoimmune diseases that may be treatedwith immunoconjugates may include acute idiopathic thrombocytopenicpurpura, chronic idiopathic thrombocytopenic purpura, dermatomyositis,Sydenham's chorea, myasthenia gravis, systemic lupus erythematosus,lupus nephritis, rheumatic fever, polyglandular syndromes, bullouspemphigoid, diabetes mellitus, Henoch-Schonlein purpura,post-streptococcal nephritis, erythema nodosum, Takayasu's arteritis,ANCA-associated vasculitides, Addison's disease, rheumatoid arthritis,multiple sclerosis, sarcoidosis, ulcerative colitis, erythemamultiforme, IgA nephropathy, polyarteritis nodosa, ankylosingspondylitis, Goodpasture's syndrome, thromboangitis obliterans,Sjogren's syndrome, primary biliary cirrhosis, Hashimoto's thyroiditis,thyrotoxicosis, scleroderma, chronic active hepatitis,polymyositis/dermatomyositis, polychondritis, bullous pemphigoid,pemphigus vulgaris, Wegener's granulomatosis, membranous nephropathy,amyotrophic lateral sclerosis, tabes dorsalis, giant cellarteritis/polymyalgia, pernicious anemia, rapidly progressiveglomerulonephritis, psoriasis or fibrosing alveolitis.

In another preferred embodiment, a therapeutic agent used in combinationwith the camptothecin conjugate of this invention may comprise one ormore isotopes. Radioactive isotopes useful for treating diseased tissueinclude, but are not limited to—¹¹¹In, ¹⁷⁷Lu, ³¹²Bi, ²¹³Bi, ²¹¹At, ⁶²Cu,⁶⁷Cu, ⁹⁰Y, ¹²⁵I, ¹³¹I, ³²P, ³³P, ⁴⁷Sc, ¹¹¹Ag, ⁶⁷Ga, ¹⁴²Pr, ¹⁵³Sm, ¹⁶¹Tb,¹⁶⁶Dy, ¹⁶⁶Ho, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁸⁹Re, ²¹²Pb, ²²³Ra, ²²⁵Ac, ⁵⁹Fe, ⁷⁵Se,⁷⁷As, ⁸⁹Sr, ⁹⁹Mo, ¹⁰⁵Rb, ¹⁰⁹Pd, ¹⁴³Pr, ¹⁴⁹Pm, ¹⁶⁹Er, ¹⁹⁴Ir, ¹⁹⁸Au,¹⁹⁹Au, ²²⁷Th and ²¹¹Pb. The therapeutic radionuclide preferably has adecay-energy in the range of 20 to 6,000 keV, preferably in the ranges60 to 200 keV for an Auger emitter, 100-2,500 keV for a beta emitter,and 4,000-6,000 keV for an alpha emitter. Maximum decay energies ofuseful beta-particle-emitting nuclides are preferably 20-5,000 keV, morepreferably 100-4,000 keV, and most preferably 500-2,500 keV. Alsopreferred are radionuclides that substantially decay with Auger-emittingparticles. For example, Co-58, Ga-67, Br-80m, Tc-99m, Rh-103m, Pt-109,In-111, Sb-119, 1-125, Ho-161, Os-189m and Ir-192. Decay energies ofuseful beta-particle-emitting nuclides are preferably <1,000 keV, morepreferably <100 keV, and most preferably <70 keV. Also preferred areradionuclides that substantially decay with generation ofalpha-particles. Such radionuclides include, but are not limited to:Dy-152, At-211, Bi-212, Ra-223, Rn-219, Po-215, Bi-211, Ac-225, Fr-221,At-217, Bi-213, Th-227 and Fm-255. Decay energies of usefulalpha-particle-emitting radionuclides are preferably 2,000-10,000 keV,more preferably 3,000-8,000 keV, and most preferably 4,000-7,000 keV.Additional potential radioisotopes of use include ¹¹C, ¹³N, ¹⁵O, ⁷⁵Br,¹⁹⁸Au, ²²⁴Ac, ¹²⁶I, ¹³³I, ⁷⁷Br, ^(113m)In, ⁹⁵Ru, ⁹⁷Ru, ¹⁰³Ru, ¹⁰⁵Ru,¹⁰⁷Hg, ²⁰³Hg, ^(121m)Te, ^(122m)Te, ^(125m)Te, ¹⁶⁵Tm, ¹⁶⁷Tm, ¹⁶⁸Tm,¹⁹⁷Pt, ¹⁰⁹Pd, ¹⁰⁵Rb, ¹⁴²Pr, ¹⁴³Pr, ¹⁶¹Tb, ¹⁶⁶Ho, ¹⁹⁹Au, ⁵⁷Co, ⁵⁸Co,⁵¹Cr, ⁵⁹Fe, ⁷⁵Se, ²⁰¹Tl, ²²⁵Ac, ⁷⁶Br, ¹⁶⁹Yb, and the like.

Radionuclides and other metals may be delivered, for example, usingchelating groups attached to an antibody or conjugate. Macrocyclicchelates such as NOTA, DOTA, and TETA are of use with a variety ofmetals and radiometals, most particularly with radionuclides of gallium,yttrium and copper, respectively. Such metal-chelate complexes can bemade very stable by tailoring the ring size to the metal of interest.Other ring-type chelates, such as macrocyclic polyethers for complexing²²³Ra, may be used.

Therapeutic agents of use in combination with the camptothecinconjugates described herein also include, for example, chemotherapeuticdrugs such as vinca alkaloids, anthracyclines, epidophyllotoxins,taxanes, antimetabolites, tyrosine kinase inhibitors, alkylating agents,antibiotics, Cox-2 inhibitors, antimitotics, antiangiogenic andproapoptotic agents, particularly doxorubicin, methotrexate, taxol,other camptothecins, and others from these and other classes ofanticancer agents, and the like. Other cancer chemotherapeutic drugsinclude nitrogen mustards, alkyl sulfonates, nitrosoureas, triazenes,folic acid analogs, pyrimidine analogs, purine analogs, platinumcoordination complexes, hormones, and the like. Suitablechemotherapeutic agents are described in REMINGTON'S PHARMACEUTICALSCIENCES, 19th Ed. (Mack Publishing Co. 1995), and in GOODMAN ANDGILMAN'S THE PHARMACOLOGICAL BASIS OF THERAPEUTICS, 7th Ed. (MacMillanPublishing Co. 1985), as well as revised editions of these publications.Other suitable chemotherapeutic agents, such as experimental drugs, areknown to those of skill in the art.

Exemplary drugs of use include, but are not limited to, 5-fluorouracil,afatinib, aplidin, azaribine, anastrozole, anthracyclines, axitinib,AVL-101, AVL-291, bendamustine, bleomycin, bortezomib, bosutinib,bryostatin-1, busulfan, calicheamycin, camptothecin, carboplatin,10-hydroxycamptothecin, carmustine, celebrex, chlorambucil, cisplatin(CDDP), Cox-2 inhibitors, irinotecan (CPT-11), SN-38, carboplatin,cladribine, camptothecans, crizotinib, cyclophosphamide, cytarabine,dacarbazine, dasatinib, dinaciclib, docetaxel, dactinomycin,daunorubicin, doxorubicin, 2-pyrrolinodoxorubicine (2P-DOX),cyano-morpholino doxorubicin, doxorubicin glucuronide, epirubicinglucuronide, erlotinib, estramustine, epidophyllotoxin, erlotinib,entinostat, estrogen receptor binding agents, etoposide (VP16),etoposide glucuronide, etoposide phosphate, exemestane, fingolimod,floxuridine (FUdR), 3′,5′-O-dioleoyl-FudR (FUdR-dO), fludarabine,flutamide, farnesyl-protein transferase inhibitors, flavopiridol,fostamatinib, ganetespib, GDC-0834, GS-1101, gefitinib, gemcitabine,hydroxyurea, ibrutinib, idarubicin, idelalisib, ifosfamide, imatinib,L-asparaginase, lapatinib, lenolidamide, leucovorin, LFM-A13, lomustine,mechlorethamine, melphalan, mercaptopurine, 6-mercaptopurine,methotrexate, mitoxantrone, mithramycin, mitomycin, mitotane, navelbine,neratinib, nilotinib, nitrosurea, olaparib, plicomycin, procarbazine,paclitaxel, PCI-32765, pentostatin, PSI-341, raloxifene, semustine,sorafenib, streptozocin, SU11248, sunitinib, tamoxifen, temazolomide (anaqueous form of DTIC), transplatinum, thalidomide, thioguanine,thiotepa, teniposide, topotecan, uracil mustard, vatalanib, vinorelbine,vinblastine, vincristine, vinca alkaloids and ZD1839. Such agents may bepart of the conjugates described herein or may alternatively beadministered in combination with the described conjugates, either priorto, simultaneously with or after the conjugate. Alternatively, one ormore therapeutic naked antibodies as are known in the art may be used incombination with the described conjugates. Exemplary therapeutic nakedantibodies are described above.

Therapeutic agents that may be used in concert with the camptothecinconjugates also may comprise toxins conjugated to targeting moieties.Toxins that may be used in this regard include ricin, abrin,ribonuclease (RNase), DNase I, Staphylococcal enterotoxin-A, pokeweedantiviral protein, gelonin, diphtheria toxin, Pseudomonas exotoxin, andPseudomonas endotoxin. (See, e.g., Pastan. et al., Cell (1986), 47:641,and Sharkey and Goldenberg, CA Cancer J Clin. 2006 July-August;56(4):226-43.) Additional toxins suitable for use herein are known tothose of skill in the art and are disclosed in U.S. Pat. No. 6,077,499.

Yet another class of therapeutic agent may comprise one or moreimmunomodulators. Immunomodulators of use may be selected from acytokine, a stem cell growth factor, a lymphotoxin, an hematopoieticfactor, a colony stimulating factor (CSF), an interferon (IFN),erythropoietin, thrombopoietin and a combination thereof. Specificallyuseful are lymphotoxins such as tumor necrosis factor (TNF),hematopoietic factors, such as interleukin (IL), colony stimulatingfactor, such as granulocyte-colony stimulating factor (G-CSF) orgranulocyte macrophage-colony stimulating factor (GM-CSF), interferon,such as interferons-α, -β, -γ or and stem cell growth factor, such asthat designated “S1 factor”. Included among the cytokines are growthhormones such as human growth hormone, N-methionyl human growth hormone,and bovine growth hormone; parathyroid hormone; thyroxine; insulin;proinsulin; relaxin; prorelaxin; glycoprotein hormones such as folliclestimulating hormone (FSH), thyroid stimulating hormone (TSH), andluteinizing hormone (LH); hepatic growth factor; prostaglandin,fibroblast growth factor; prolactin; placental lactogen, OB protein;tumor necrosis factor-α and -β; mullerian-inhibiting substance; mousegonadotropin-associated peptide; inhibin; activin; vascular endothelialgrowth factor; integrin; thrombopoietin (TPO); nerve growth factors suchas NGF-β; platelet-growth factor; transforming growth factors (TGFs)such as TGF-α and TGF-β; insulin-like growth factor-I and -II;erythropoietin (EPO); osteoinductive factors; interferons such asinterferon-α, -β, and -γ; colony stimulating factors (CSFs) such asmacrophage-CSF (M-CSF); interleukins (ILs) such as IL-1, IL-1α, IL-2,IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-13,IL-14, IL-15, IL-16, IL-17, IL-18, IL-21, IL-25, LIF, kit-ligand orFLT-3, angiostatin, thrombospondin, endostatin, tumor necrosis factorand lymphotoxin (LT). As used herein, the term cytokine includesproteins from natural sources or from recombinant cell culture andbiologically active equivalents of the native sequence cytokines.

Chemokines of use include RANTES, MCAF, MIP1-alpha, MIP1-Beta and IP-10.

The person of ordinary skill will realize that the subjectimmunoconjugates, comprising a camptothecin conjugated to an antibody orantibody fragment, may be used alone or in combination with one or moreother therapeutic agents, such as a second antibody, second antibodyfragment, second immunoconjugate, radionuclide, toxin, drug,chemotherapeutic agent, radiation therapy, chemokine, cytokine,immunomodulator, enzyme, hormone, oligonucleotide, RNAi or siRNA. Suchadditional therapeutic agents may be administered separately, incombination with, or attached to the subject antibody-drugimmunoconjugates.

Formulation and Administration

Suitable routes of administration of the conjugates include, withoutlimitation, oral, parenteral, subcutaneous, rectal, transmucosal,intestinal administration, intramuscular, intramedullary, intrathecal,direct intraventricular, intravenous, intravitreal, intraperitoneal,intranasal, or intraocular injections. The preferred routes ofadministration are parenteral. Alternatively, one may administer thecompound in a local rather than systemic manner, for example, viainjection of the compound directly into a solid tumor.

Immunoconjugates can be formulated according to known methods to preparepharmaceutically useful compositions, whereby the immunoconjugate iscombined in a mixture with a pharmaceutically suitable excipient.Sterile phosphate-buffered saline is one example of a pharmaceuticallysuitable excipient. Other suitable excipients are well-known to those inthe art. See, for example, Ansel et al., PHARMACEUTICAL DOSAGE FORMS ANDDRUG DELIVERY SYSTEMS, 5th Edition (Lea & Febiger 1990), and Gennaro(ed.), REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Edition (MackPublishing Company 1990), and revised editions thereof.

In a preferred embodiment, the immunoconjugate is formulated in Good'sbiological buffer (pH 6-7), using a buffer selected from the groupconsisting of N-(2-acetamido)-2-aminoethanesulfonic acid (ACES);N-(2-acetamido)iminodiacetic acid (ADA);N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES);4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES);2-(N-morpholino)ethanesulfonic acid (MES);3-(N-morpholino)propanesulfonic acid (MOPS);3-(N-morpholinyl)-2-hydroxypropanesulfonic acid (MOPSO); andpiperazine-N,N′-bis(2-ethanesulfonic acid) [Pipes]. More preferredbuffers are IVIES or MOPS, preferably in the concentration range of 20to 100 mM, more preferably about 25 mM. Most preferred is 25 mM MES, pH6.5. The formulation may further comprise 25 mM trehalose and 0.01% v/vpolysorbate 80 as excipients, with the final buffer concentrationmodified to 22.25 mM as a result of added excipients. The preferredmethod of storage is as a lyophilized formulation of the conjugates,stored in the temperature range of −20° C. to 2° C., with the mostpreferred storage at 2° C. to 8° C.

The immunoconjugate can be formulated for intravenous administrationvia, for example, bolus injection, slow infusion or continuous infusion.Preferably, the antibody of the present invention is infused over aperiod of less than about 4 hours, and more preferably, over a period ofless than about 3 hours. For example, the first 25-50 mg could beinfused within 30 minutes, preferably even 15 min, and the remainderinfused over the next 2-3 hrs. Formulations for injection can bepresented in unit dosage form, e.g., in ampoules or in multi-dosecontainers, with an added preservative. The compositions can take suchforms as suspensions, solutions or emulsions in oily or aqueousvehicles, and can contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient can be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

Additional pharmaceutical methods may be employed to control theduration of action of the therapeutic conjugate. Control releasepreparations can be prepared through the use of polymers to complex oradsorb the immunoconjugate. For example, biocompatible polymers includematrices of poly(ethylene-co-vinyl acetate) and matrices of apolyanhydride copolymer of a stearic acid dimer and sebacic acid.Sherwood et al., Bio/Technology 10: 1446 (1992). The rate of release ofan immunoconjugate from such a matrix depends upon the molecular weightof the immunoconjugate, the amount of immunoconjugate within the matrix,and the size of dispersed particles. Saltzman et al., Biophys. J. 55:163 (1989); Sherwood et al., supra. Other solid dosage forms aredescribed in Ansel et al., PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERYSYSTEMS, 5th Edition (Lea & Febiger 1990), and Gennaro (ed.),REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Edition (Mack PublishingCompany 1990), and revised editions thereof.

Generally, the dosage of an administered immunoconjugate for humans willvary depending upon such factors as the patient's age, weight, height,sex, general medical condition and previous medical history. It may bedesirable to provide the recipient with a dosage of immunoconjugate thatis in the range of from about 1 mg/kg to 24 mg/kg as a singleintravenous infusion, although a lower or higher dosage also may beadministered as circumstances dictate. A dosage of 1-20 mg/kg for a 70kg patient, for example, is 70-1,400 mg, or 41-824 mg/m² for a 1.7-mpatient. The dosage may be repeated as needed, for example, once perweek for 4-10 weeks, once per week for 8 weeks, or once per week for 4weeks. It may also be given less frequently, such as every other weekfor several months, or monthly or quarterly for many months, as neededin a maintenance therapy. Preferred dosages may include, but are notlimited to, 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 7mg/kg, 8 mg/kg, 9 mg/kg, 10 mg/kg, 11 mg/kg, 12 mg/kg, 13 mg/kg, 14mg/kg, 15 mg/kg, 16 mg/kg, 17 mg/kg, 18 mg/kg, 19 mg/kg, 20 mg/kg, 22mg/kg and 24 mg/kg. Any amount in the range of 1 to 24 mg/kg may beused. The dosage is preferably administered multiple times, once ortwice a week. A minimum dosage schedule of 4 weeks, more preferably 8weeks, more preferably 16 weeks or longer may be used. The schedule ofadministration may comprise administration once or twice a week, on acycle selected from the group consisting of: (i) weekly; (ii) everyother week; (iii) one week of therapy followed by two, three or fourweeks off; (iv) two weeks of therapy followed by one, two, three or fourweeks off; (v) three weeks of therapy followed by one, two, three, fouror five week off; (vi) four weeks of therapy followed by one, two,three, four or five week off; (vii) five weeks of therapy followed byone, two, three, four or five week off; and (viii) monthly. The cyclemay be repeated 4, 6, 8, 10, 12, 16 or 20 times or more.

Alternatively, an immunoconjugate may be administered as one dosageevery 2 or 3 weeks, repeated for a total of at least 3 dosages. Or,twice per week for 4-6 weeks. If the dosage is lowered to approximately200-300 mg/m² (340 mg per dosage for a 1.7-m patient, or 4.9 mg/kg for a70 kg patient), it may be administered once or even twice weekly for 4to 10 weeks. Alternatively, the dosage schedule may be decreased, namelyevery 2 or 3 weeks for 2-3 months. It has been determined, however, thateven higher doses, such as 12 mg/kg once weekly or once every 2-3 weekscan be administered by slow i.v. infusion, for repeated dosing cycles.The dosing schedule can optionally be repeated at other intervals anddosage may be given through various parenteral routes, with appropriateadjustment of the dose and schedule.

In preferred embodiments, the immunoconjugates are of use for therapy ofcancer. Examples of cancers include, but are not limited to, carcinoma,lymphoma, glioblastoma, melanoma, sarcoma, and leukemia, myeloma, orlymphoid malignancies. More particular examples of such cancers arenoted below and include: squamous cell cancer (e.g., epithelial squamouscell cancer), Ewing sarcoma, Wilms tumor, astrocytomas, lung cancerincluding small-cell lung cancer, non-small cell lung cancer,adenocarcinoma of the lung and squamous carcinoma of the lung, cancer ofthe peritoneum, gastric or stomach cancer including gastrointestinalcancer, pancreatic cancer, glioblastoma multiforme, cervical cancer,ovarian cancer, liver cancer, bladder cancer, hepatoma, hepatocellularcarcinoma, neuroendocrine tumors, medullary thyroid cancer,differentiated thyroid carcinoma, breast cancer, ovarian cancer, coloncancer, rectal cancer, endometrial cancer or uterine carcinoma, salivarygland carcinoma, kidney or renal cancer, prostate cancer, vulvar cancer,anal carcinoma, penile carcinoma, as well as head-and-neck cancer. Theterm “cancer” includes primary malignant cells or tumors (e.g., thosewhose cells have not migrated to sites in the subject's body other thanthe site of the original malignancy or tumor) and secondary malignantcells or tumors (e.g., those arising from metastasis, the migration ofmalignant cells or tumor cells to secondary sites that are differentfrom the site of the original tumor).

Other examples of cancers or malignancies include, but are not limitedto: Acute Childhood Lymphoblastic Leukemia, Acute LymphoblasticLeukemia, Acute Lymphocytic Leukemia, Acute Myeloid Leukemia,Adrenocortical Carcinoma, Adult (Primary) Hepatocellular Cancer, Adult(Primary) Liver Cancer, Adult Acute Lymphocytic Leukemia, Adult AcuteMyeloid Leukemia, Adult Hodgkin's Lymphoma, Adult Lymphocytic Leukemia,Adult Non-Hodgkin's Lymphoma, Adult Primary Liver Cancer, Adult SoftTissue Sarcoma, AIDS-Related Lymphoma, AIDS-Related Malignancies, AnalCancer, Astrocytoma, Bile Duct Cancer, Bladder Cancer, Bone Cancer,Brain Stem Glioma, Brain Tumors, Breast Cancer, Cancer of the RenalPelvis and Ureter, Central Nervous System (Primary) Lymphoma, CentralNervous System Lymphoma, Cerebellar Astrocytoma, Cerebral Astrocytoma,Cervical Cancer, Childhood (Primary) Hepatocellular Cancer, Childhood(Primary) Liver Cancer, Childhood Acute Lymphoblastic Leukemia,Childhood Acute Myeloid Leukemia, Childhood Brain Stem Glioma, ChildhoodCerebellar Astrocytoma, Childhood Cerebral Astrocytoma, ChildhoodExtracranial Germ Cell Tumors, Childhood Hodgkin's Disease, ChildhoodHodgkin's Lymphoma, Childhood Hypothalamic and Visual Pathway Glioma,Childhood Lymphoblastic Leukemia, Childhood Medulloblastoma, ChildhoodNon-Hodgkin's Lymphoma, Childhood Pineal and Supratentorial PrimitiveNeuroectodermal Tumors, Childhood Primary Liver Cancer, ChildhoodRhabdomyosarcoma, Childhood Soft Tissue Sarcoma, Childhood VisualPathway and Hypothalamic Glioma, Chronic Lymphocytic Leukemia, ChronicMyelogenous Leukemia, Colon Cancer, Cutaneous T-Cell Lymphoma, EndocrinePancreas Islet Cell Carcinoma, Endometrial Cancer, Ependymoma,Epithelial Cancer, Esophageal Cancer, Ewing's Sarcoma and RelatedTumors, Exocrine Pancreatic Cancer, Extracranial Germ Cell Tumor,Extragonadal Germ Cell Tumor, Extrahepatic Bile Duct Cancer, Eye Cancer,Female Breast Cancer, Gaucher's Disease, Gallbladder Cancer, GastricCancer, Gastrointestinal Carcinoid Tumor, Gastrointestinal Tumors, GermCell Tumors, Gestational Trophoblastic Tumor, Hairy Cell Leukemia, Headand Neck Cancer, Hepatocellular Cancer, Hodgkin's Lymphoma,Hypergammaglobulinemia, Hypopharyngeal Cancer, Intestinal Cancers,Intraocular Melanoma, Islet Cell Carcinoma, Islet Cell PancreaticCancer, Kaposi's Sarcoma, Kidney Cancer, Laryngeal Cancer, Lip and OralCavity Cancer, Liver Cancer, Lung Cancer, Lymphoproliferative Disorders,Macroglobulinemia, Male Breast Cancer, Malignant Mesothelioma, MalignantThymoma, Medulloblastoma, Melanoma, Mesothelioma, Metastatic OccultPrimary Squamous Neck Cancer, Metastatic Primary Squamous Neck Cancer,Metastatic Squamous Neck Cancer, Multiple Myeloma, MultipleMyeloma/Plasma Cell Neoplasm, Myelodysplastic Syndrome, MyelogenousLeukemia, Myeloid Leukemia, Myeloproliferative Disorders, Nasal Cavityand Paranasal Sinus Cancer, Nasopharyngeal Cancer, Neuroblastoma,Non-Hodgkin's Lymphoma, Nonmelanoma Skin Cancer, Non-Small Cell LungCancer, Occult Primary Metastatic Squamous Neck Cancer, OropharyngealCancer, Osteo-/Malignant Fibrous Sarcoma, Osteosarcoma/Malignant FibrousHistiocytoma, Osteosarcoma/Malignant Fibrous Histiocytoma of Bone,Ovarian Epithelial Cancer, Ovarian Germ Cell Tumor, Ovarian LowMalignant Potential Tumor, Pancreatic Cancer, Paraproteinemias,Polycythemia vera, Parathyroid Cancer, Penile Cancer, Pheochromocytoma,Pituitary Tumor, Primary Central Nervous System Lymphoma, Primary LiverCancer, Prostate Cancer, Rectal Cancer, Renal Cell Cancer, Renal Pelvisand Ureter Cancer, Retinoblastoma, Rhabdomyosarcoma, Salivary GlandCancer, Sarcoidosis Sarcomas, Sezary Syndrome, Skin Cancer, Small CellLung Cancer, Small Intestine Cancer, Soft Tissue Sarcoma, Squamous NeckCancer, Stomach Cancer, Supratentorial Primitive Neuroectodermal andPineal Tumors, T-Cell Lymphoma, Testicular Cancer, Thymoma, ThyroidCancer, Transitional Cell Cancer of the Renal Pelvis and Ureter,Transitional Renal Pelvis and Ureter Cancer, Trophoblastic Tumors,Ureter and Renal Pelvis Cell Cancer, Urethral Cancer, Uterine Cancer,Uterine Sarcoma, Vaginal Cancer, Visual Pathway and Hypothalamic Glioma,Vulvar Cancer, Waldenstrom's macroglobulinemia, Wilms' tumor, and anyother hyperproliferative disease, besides neoplasia, located in an organsystem listed above.

The methods and compositions described and claimed herein may be used totreat malignant or premalignant conditions and to prevent progression toa neoplastic or malignant state, including but not limited to thosedisorders described above. Such uses are indicated in conditions knownor suspected of preceding progression to neoplasia or cancer, inparticular, where non-neoplastic cell growth consisting of hyperplasia,metaplasia, or most particularly, dysplasia has occurred (for review ofsuch abnormal growth conditions, see Robbins and Angell, BasicPathology, 2d Ed., W. B. Saunders Co., Philadelphia, pp. 68-79 (1976)).

Dysplasia is frequently a forerunner of cancer, and is found mainly inthe epithelia. It is the most disorderly form of non-neoplastic cellgrowth, involving a loss in individual cell uniformity and in thearchitectural orientation of cells. Dysplasia characteristically occurswhere there exists chronic irritation or inflammation. Dysplasticdisorders which can be treated include, but are not limited to,anhidrotic ectodermal dysplasia, anterofacial dysplasia, asphyxiatingthoracic dysplasia, atriodigital dysplasia, bronchopulmonary dysplasia,cerebral dysplasia, cervical dysplasia, chondroectodermal dysplasia,cleidocranial dysplasia, congenital ectodermal dysplasia,craniodiaphysial dysplasia, craniocarpotarsal dysplasia,craniometaphysial dysplasia, dentin dysplasia, diaphysial dysplasia,ectodermal dysplasia, enamel dysplasia, encephalo-ophthalmic dysplasia,dysplasia epiphysialis hemimelia, dysplasia epiphysialis multiplex,dysplasia epiphysialis punctata, epithelial dysplasia,faciodigitogenital dysplasia, familial fibrous dysplasia of jaws,familial white folded dysplasia, fibromuscular dysplasia, fibrousdysplasia of bone, florid osseous dysplasia, hereditary renal-retinaldysplasia, hidrotic ectodermal dysplasia, hypohidrotic ectodermaldysplasia, lymphopenic thymic dysplasia, mammary dysplasia,mandibulofacial dysplasia, metaphysial dysplasia, Mondini dysplasia,monostotic fibrous dysplasia, mucoepithelial dysplasia, multipleepiphysial dysplasia, oculoauriculovertebral dysplasia,oculodentodigital dysplasia, oculovertebral dysplasia, odontogenicdysplasia, opthalmomandibulomelic dysplasia, periapical cementaldysplasia, polyostotic fibrous dysplasia, pseudoachondroplasticspondyloepiphysial dysplasia, retinal dysplasia, septo-optic dysplasia,spondyloepiphysial dysplasia, and ventriculoradial dysplasia.

Additional pre-neoplastic disorders which can be treated include, butare not limited to, benign dysproliferative disorders (e.g., benigntumors, fibrocystic conditions, tissue hypertrophy, intestinal polyps oradenomas, and esophageal dysplasia), leukoplakia, keratoses, Bowen'sdisease, Farmer's Skin, solar cheilitis, and solar keratosis.

In preferred embodiments, the method of the invention is used to inhibitgrowth, progression, and/or metastasis of cancers, in particular thoselisted above.

Additional hyperproliferative diseases, disorders, and/or conditionsinclude, but are not limited to, progression, and/or metastases ofmalignancies and related disorders such as leukemia (including acuteleukemias; e.g., acute lymphocytic leukemia, acute myelocytic leukemia[including myeloblastic, promyelocytic, myelomonocytic, monocytic, anderythroleukemia]) and chronic leukemias (e.g., chronic myelocytic[granulocytic] leukemia and chronic lymphocytic leukemia), polycythemiavera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease),multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease,and solid tumors including, but not limited to, sarcomas and carcinomassuch as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma,osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma,lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma,Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma,pancreatic cancer, breast cancer, ovarian cancer, prostate cancer,squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweatgland carcinoma, sebaceous gland carcinoma, papillary carcinoma,papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma,bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile ductcarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor,cervical cancer, testicular tumor, lung carcinoma, small cell lungcarcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,emangioblastoma, acoustic neuroma, oligodendroglioma, meningioma,melanoma, neuroblastoma, and retinoblastoma.

Autoimmune diseases that may be treated with immunoconjugates mayinclude acute and chronic immune thrombocytopenias, dermatomyositis,Sydenham's chorea, myasthenia gravis, systemic lupus erythematosus,lupus nephritis, rheumatic fever, polyglandular syndromes, bullouspemphigoid, diabetes mellitus, Henoch-Schonlein purpura,post-streptococcal nephritis, erythema nodosum, Takayasu's arteritis,ANCA-associated vasculitides, Addison's disease, rheumatoid arthritis,multiple sclerosis, sarcoidosis, ulcerative colitis, erythemamultiforme, IgA nephropathy, polyarteritis nodosa, ankylosingspondylitis, Goodpasture's syndrome, thromboangitis obliterans,Sjogren's syndrome, primary biliary cirrhosis, Hashimoto's thyroiditis,thyrotoxicosis, scleroderma, chronic active hepatitis,polymyositis/dermatomyositis, polychondritis, bullous pemphigoid,pemphigus vulgaris, Wegener's granulomatosis, membranous nephropathy,amyotrophic lateral sclerosis, tabes dorsalis, giant cellarteritis/polymyalgia, pernicious anemia, rapidly progressiveglomerulonephritis, psoriasis or fibrosing alveolitis.

Kits

Various embodiments may concern kits containing components suitable fortreating diseased tissue in a patient. Exemplary kits may contain atleast one conjugated antibody or other targeting moiety as describedherein. If the composition containing components for administration isnot formulated for delivery via the alimentary canal, such as by oraldelivery, a device capable of delivering the kit components through someother route may be included. One type of device, for applications suchas parenteral delivery, is a syringe that is used to inject thecomposition into the body of a subject. Inhalation devices may also beused.

The kit components may be packaged together or separated into two ormore containers. In some embodiments, the containers may be vials thatcontain sterile, lyophilized formulations of a composition that aresuitable for reconstitution. A kit may also contain one or more bufferssuitable for reconstitution and/or dilution of other reagents. Othercontainers that may be used include, but are not limited to, a pouch,tray, box, tube, or the like. Kit components may be packaged andmaintained sterilely within the containers. Another component that canbe included is instructions to a person using a kit for its use.

EXAMPLES

Various embodiments of the present invention are illustrated by thefollowing examples, without limiting the scope thereof.

Example 1. Production and Use of Anti-Trop-2-SN-38 Antibody-DrugConjugate

The humanized RS7 (hRS7) anti-Trop-2 antibody was produced as describedin U.S. Pat. No. 7,238,785, the Figures and Examples section of whichare incorporated herein by reference. SN-38 attached to a CL2A linkerwas produced and conjugated to hRS7 (anti-Trop-2), hPAM4 (anti-MUC5ac),hA20 (anti-CD20) or hMN-14 (anti-CEACAM5) antibodies according to U.S.Pat. No. 7,999,083 (Example 10 and 12 of which are incorporated hereinby reference). The conjugation protocol resulted in a ratio of about 6SN-38 molecules attached per antibody molecule.

Immune-compromised athymic nude mice (female), bearing subcutaneoushuman pancreatic or colon tumor xenografts were treated with eitherspecific CL2A-SN-38 conjugate or control conjugate or were leftuntreated. The therapeutic efficacies of the specific conjugates wereobserved. FIG. 1 shows a Capan 1 pancreatic tumor model, whereinspecific CL2A-SN-38 conjugates of hRS7 (anti-Trop-2), hPAM4(anti-MUC-5ac), and hMN-14 (anti-CEACAM5) antibodies showed betterefficacies than control hA20-CL2A-SN-38 conjugate (anti-CD20) anduntreated control. Similarly in a BXPC3 model of human pancreaticcancer, the specific hRS7-CL2A-SN-38 showed better therapeutic efficacythan control treatments (FIG. 2).

Example 2. ADCC Activity of Anti-Trop-2 ADCs

The ADCC activity of various hRS7-ADC conjugates was determined incomparison to hRS7 IgG (FIG. 3). PBMCs were purified from bloodpurchased from the Blood Center of New Jersey. A Trop-2-positive humanpancreatic adenocarcinoma cell line (BxPC-3) was used as the target cellline with an effector to target ratio of 100:1. ADCC mediated by hRS7IgG was compared to hRS7-Pro-2-PDox, hRS7-CL2A-SN-38, and the reducedand capped hRS7-NEM. All were used at 33.3 nM.

Results are shown in FIG. 3. Overall activity was low, but significant.There was 8.5% specific lysis for the hRS7 IgG which was notsignificantly different from hRS7-Pro-2-PDox. Both were significantlybetter than hLL2 control and hRS7-NEM and hRS7-SN-38 (P<0.02, two-tailedt-test). There was no difference between hRS7-NEM and hRS7-SN-38.

Example 3. Efficacy of Anti-Trop-2-SN-38 ADC Against Diverse EpithelialCancers In Vivo

Abstract

The purpose of this study was to evaluate the efficacy of anSN-38-anti-Trop-2 (hRS7) ADC against several human solid tumor types,and to assess its tolerability in mice and monkeys, the latter withtissue cross-reactivity to hRS7 similar to humans. Two SN-38derivatives, CL2-SN-38 and CL2A-SN-38, were conjugated to theanti-Trop-2-humanized antibody, hRS7. The immunoconjugates werecharacterized in vitro for stability, binding, and cytotoxicity.Efficacy was tested in five different human solid tumor-xenograft modelsthat expressed Trop-2 antigen. Toxicity was assessed in mice and inCynomolgus monkeys.

The hRS7 conjugates of the two SN-38 derivatives were equivalent in drugsubstitution (˜6), cell binding (K_(d)˜1.2 nmol/L), cytotoxicity(IC₅₀˜2.2 nmol/L), and serum stability in vitro (t/_(1/2)˜20 hours).Exposure of cells to the ADC demonstrated signaling pathways leading toPARP cleavage, but differences versus free SN-38 in p53 and p21upregulation were noted. Significant antitumor effects were produced byhRS7-SN-38 at nontoxic doses in mice bearing Calu-3 (P≤0.05), Capan-1(P<0.018), BxPC-3 (P<0.005), and COLO 205 tumors (P<0.033) when comparedto nontargeting control ADCs. Mice tolerated a dose of 2×12 mg/kg (SN-38equivalents) with only short-lived elevations in ALT and AST liverenzyme levels. Cynomolgus monkeys infused with 2×0.96 mg/kg exhibitedonly transient decreases in blood counts, although, importantly, thevalues did not fall below normal ranges.

In summary, the anti-Trop-2 hRS7-CL2A-SN-38 ADC provided significant andspecific antitumor effects against a range of human solid tumor types.It was well tolerated in monkeys, with tissue Trop-2 expression similarto humans, at clinically relevant doses.

INTRODUCTION

Successful irinotecan treatment of patients with solid tumors has beenlimited, due in large part to the low conversion rate of the CPT-11prodrug into the active SN-38 metabolite. Others have examinednontargeted forms of SN-38 as a means to bypass the need for thisconversion and to deliver SN-38 passively to tumors. We conjugated SN-38covalently to a humanized anti-Trop-2 antibody, hRS7. This antibody-drugconjugate has specific antitumor effects in a range of s.c. human cancerxenograft models, including non-small cell lung carcinoma, pancreatic,colorectal, and squamous cell lung carcinomas, all at nontoxic doses(e.g., ≤3.2 mg/kg cumulative SN-38 equivalent dose). Trop-2 is widelyexpressed in many epithelial cancers, but also some normal tissues, andtherefore a dose escalation study in Cynomolgus monkeys was performed toassess the clinical safety of this conjugate. Monkeys tolerated 24 mgSN-38 equivalents/kg with only minor, reversible, toxicities. Given itstumor-targeting and safety profile, hRS7-SN-38 provides a significantimprovement in the management of solid tumors responsive to irinotecan.

Material and Methods

Cell lines, antibodies, and chemotherapeutics—All human cancer celllines used in this study were purchased from the American Type CultureCollection. These include Calu-3 (non-small cell lung carcinoma),SK-MES-1 (squamous cell lung carcinoma), COLO 205 (colonicadenocarcinoma), Capan-1 and BxPC-3 (pancreatic adenocarcinomas), andPC-3 (prostatic adenocarcinomas). Humanized RS7 IgG and controlhumanized anti-CD20 (hA20 IgG, veltuzumab) and anti-CD22 (hLL2 IgG,epratuzumab) antibodies were prepared at Immunomedics, Inc. Irinotecan(20 mg/mL) was obtained from Hospira, Inc.

SN-38 immunoconjugates and in vitro aspects—Synthesis of CL2-SN-38 hasbeen described previously (Moon et al., 2008, J Med Chem 51:6916-26).Its conjugation to hRS7 IgG and serum stability were performed asdescribed (Moon et al., 2008, J Med Chem 51:6916-26; Govindan et al.,2009, Clin Chem Res 15:6052-61). Preparations of CL2A-SN-38 (M.W. 1480)and its hRS7 conjugate, and stability, binding, and cytotoxicitystudies, were conducted as described in the preceding Examples.

In vivo therapeutic studies—For all animal studies, the doses of SN-38immunoconjugates and irinotecan are shown in SN-38 equivalents. Based ona mean SN-38/IgG substitution ratio of 6, a dose of 500 μg ADC to a 20-gmouse (25 mg/kg) contains 0.4 mg/kg of SN-38. Irinotecan doses arelikewise shown as SN-38 equivalents (i.e., 40 mg irinotecan/kg isequivalent to 24 mg/kg of SN-38).

NCr female athymic nude (nu/nu) mice, 4 to 8 weeks old, and maleSwiss-Webster mice, 10 weeks old, were purchased from Taconic Farms.Tolerability studies were performed in Cynomolgus monkeys (Macacafascicularis; 2.5-4 kg male and female) by SNBL USA, Ltd. Animals wereimplanted subcutaneously with different human cancer cell lines. Tumorvolume (TV) was determined by measurements in 2 dimensions usingcalipers, with volumes defined as: L×w²/2, where L is the longestdimension of the tumor and w is the shortest. Tumors ranged in sizebetween 0.10 and 0.47 cm³ when therapy began. Treatment regimens,dosages, and number of animals in each experiment are described in theResults. The lyophilized hRS7-CL2A-SN-38 and control ADC werereconstituted and diluted as required in sterile saline. All reagentswere administered intraperitoneally (0.1 mL), except irinotecan, whichwas administered intravenously. The dosing regimen was influenced by ourprior investigations, where the ADC was given every 4 days or twiceweekly for varying lengths of time (Moon et al., 2008, J Med Chem51:6916-26; Govindan et al., 2009, Clin Chem Res 15:6052-61). Thisdosing frequency reflected a consideration of the conjugate's serumhalf-life in vitro, to allow a more continuous exposure to the ADC.

Statistics—Growth curves are shown as percent change in initial TV overtime. Statistical analysis of tumor growth was based on area under thecurve (AUC). Profiles of individual tumor growth were obtained throughlinear-curve modeling. Anf-test was employed to determine equality ofvariance between groups before statistical analysis of growth curves. A2-tailed t-test was used to assess statistical significance between thevarious treatment groups and controls, except for the saline control,where a 1-tailed t-test was used (significance at P<0.05). Statisticalcomparisons of AUC were performed only up to the time that the firstanimal within a group was euthanized due to progression.

Pharmacokinetics and biodistribution—¹¹¹In-radiolabeled hRS7-CL2A-SN-38and hRS7 IgG were injected into nude mice bearing s.c. SK-MES-1 tumors(˜0.3 cm³). One group was injected intravenously with 20 μCi (250-μgprotein) of ¹¹¹In-hRS7-CL2A-SN-38, whereas another group received 20 μCi(250-μg protein) of ¹¹¹In-hRS7 IgG. At various timepoints mice (5 pertimepoint) were anesthetized, bled via intracardiac puncture, and theneuthanized. Tumors and various tissues were removed, weighed, andcounted by γ scintillation to determine the percentage injected dose pergram tissue (% ID/g). A third group was injected with 250 μg ofunlabeled hRS7-CL2A-SN-38 3 days before the administration of¹¹¹In-hRS7-CL2A-SN-38 and likewise necropsied. A 2-tailed t-test wasused to compare hRS7-CL2A-SN-38 and hRS7 IgG uptake after determiningequality of variance using thef-test. Pharmacokinetic analysis on bloodclearance was performed using WinNonLin software (Parsight Corp.).

Tolerability in Swiss-Webster mice and Cynomolgus monkeys—Briefly, micewere sorted into 4 groups each to receive 2-mL i.p. injections of eithera sodium acetate buffer control or 3 different doses of hRS7-CL2A-SN-38(4, 8, or 12 mg/kg of SN-38) on days 0 and 3 followed by blood and serumcollection, as described in Results. Cynomolgus monkeys (3 male and 3female; 2.5-4.0 kg) were administered 2 different doses ofhRS7-CL2A-SN-38. Dosages, times, and number of monkeys bled forevaluation of possible hematologic toxicities and serum chemistries aredescribed in the Results.

Results

Stability and potency of hRS7-CL2A-SN-38—Two different linkages wereused to conjugate SN-38 to hRS7 IgG (FIG. 4 (A)). The first is termedCL2-SN-38 and has been described previously (Moon et al., 2008, J MedChem 51:6916-26; Govindan et al., 2009, Clin Chem Res 15:6052-61). Achange in the synthesis of CL2 to remove the phenylalanine moiety withinthe linker was used to produce the CL2A linker. This change simplifiedthe synthesis, but did not affect the conjugation outcome (e.g., bothCL2-SN-38 and CL2A-SN-38 incorporated ˜6 SN-38 per IgG molecule).Side-by-side comparisons found no significant differences in serumstability, antigen binding, or in vitro cytotoxicity. This result wassurprising, since the phenylalanine residue in CL2 is part of a designedcleavage site for cathepsin B, a lysosomal protease.

To confirm that the change in the SN-38 linker from CL2 to CL2A did notimpact in vivo potency, hRS7-CL2A and hRS7-CL2-SN-38 were compared inmice bearing COLO 205 (FIG. 4 (B)) or Capan-1 tumors (FIG. 4 (C)), using0.4 mg or 0.2 mg/kg SN-38 twice weekly×4 weeks, respectively, and withstarting tumors of 0.25 cm³ size in both studies. Both the hRS7-CL2A andCL2-SN-38 conjugates significantly inhibited tumor growth compared tountreated (AUC_(14days) P<0.002 vs. saline in COLO 205 model;AUC_(21days) P<0.001 vs. saline in Capan-1 model), and a nontargetinganti-CD20 control ADC, hA20-CL2A-SN-38 (AUC_(14days) P<0.003 in COLO-205model; AUC_(35days): P<0.002 in Capan-1 model). At the end of the study(day 140) in the Capan-1 model, 50% of the mice treated withhRS7-CL2A-SN-38 and 40% of the hRS7-CL2-SN-38 mice were tumor-free,whereas only 20% of the hA20-ADC-treated animals had no visible sign ofdisease. As demonstrated in FIG. 4, the CL2A linker resulted in asomewhat higher efficacy compared to CL2.

Mechanism of action—In vitro cytotoxicity studies demonstrated thathRS7-CL2A-SN-38 had IC₅₀ values in the nmol/L range against severaldifferent solid tumor lines (Table 6). The IC₅₀ with free SN-38 waslower than the conjugate in all cell lines. Although there was noapparent correlation between Trop-2 expression and sensitivity tohRS7-CL2A-SN-38, the IC₅₀ ratio of the ADC versus free SN-38 was lowerin the higher Trop-2-expressing cells, most likely reflecting theenhanced ability to internalize the drug when more antigen is present.

SN-38 is known to activate several signaling pathways in cells, leadingto apoptosis (e.g., Cusack et al., 2001, Cancer Res 61:3535-40; Liu etal. 2009, Cancer Lett 274:47-53; Lagadec et al., 2008, Br J Cancer98:335-44). Our initial studies examined the expression of 2 proteinsinvolved in early signaling events (p21^(Waf1/Cip1) and p53) and 1 lateapoptotic event [cleavage of poly-ADP-ribose polymerase (PARP)] in vitro(not shown). In BxPC-3, SN-38 led to a 20-fold increase inp21^(Waf1/Cip1) expression (not shown), whereas hRS7-CL2A-SN-38 resultedin only a 10-fold increase (not shown), a finding consistent with thehigher activity with free SN-38 in this cell line (Table 6). However,hRS7-CL2A-SN-38 increased p21^(Waf1/Cip1) expression in Calu-3 more than2-fold over free SN-38 (not shown).

A greater disparity between hRS7-CL2A-SN-38- and free SN-38-mediatedsignaling events was observed in p53 expression (not shown). In bothBxPC-3 and Calu-3, upregulation of p53 with free SN-38 was not evidentuntil 48 hours, whereas hRS7-CL2A-SN-38 upregulated p53 within 24 hours(not shown). In addition, p53 expression in cells exposed to the ADC washigher in both cell lines compared to SN-38 (not shown). Interestingly,although hRS7 IgG had no appreciable effect on p21^(Waf1/Cip1)expression, it did induce the upregulation of p53 in both BxPC-3 andCalu-3, but only after a 48-hour exposure (not shown). In terms of laterapoptotic events, cleavage of PARP was evident in both cell lines whenincubated with either SN-38 or the conjugate (not shown). The presenceof the cleaved PARP was higher at 24 hours in BxPC-3 (not shown), whichcorrelates with high expression of p21 and its lower IC₅₀. The higherdegree of cleavage with free SN-38 over the ADC was consistent with thecytotoxicity findings.

Efficacy of hRS7-SN-38—Because Trop-2 is widely expressed in severalhuman carcinomas, studies were performed in several different humancancer models, which started using the hRS7-CL2-SN-38 linkage, butlater, conjugates with the CL2A-linkage were used. Calu-3-bearing nudemice given 0.04 mg SN-38/kg of the hRS7-CL2-SN-38 every 4 days×4 had asignificantly improved response compared to animals administered theequivalent amount of non-targeting hLL2-CL2-SN-38 (TV=0.14±0.22 cm³ vs.0.80±0.91 cm³, respectively; AUC_(42days) P<0.026; FIG. 5A). Adose-response was observed when the dose was increased to 0.4 mg/kgSN-38 (FIG. 5A). At this higher dose level, all mice given the specifichRS7 conjugate were “cured” within 28 days, and remained tumor-freeuntil the end of the study on day 147, whereas tumors regrew in animalstreated with the irrelevant ADC (specific vs. irrelevant AUC_(98days):P=0.05). In mice receiving the mixture of hRS7 IgG and SN-38, tumorsprogressed >4.5-fold by day 56 (TV=1.10±0.88 cm³; AUC_(56 days) P<0.006vs. hRS7-CL2-SN-38) (FIG. 5A).

Efficacy also was examined in human colonic (COLO 205) and pancreatic(Capan-1) tumor xenografts. In COLO 205 tumor-bearing animals, (FIG.5B), hRS7-CL2-SN-38 (0.4 mg/kg, q4d×8) prevented tumor growth over the28-day treatment period with significantly smaller tumors compared tocontrol anti-CD20 ADC (hA20-CL2-SN-38), or hRS7 IgG (TV=0.16±0.09 cm³,1.19±0.59 cm³, and 1.77±0.93 cm³, respectively; AUC_(28days) P<0.016).

TABLE 6 Expression of Trop-2 in vitro cytotoxicity of SN-38 andhRS7-SN-38 in various solid tumor lines Cytotoxicity results hRS7-SN-Trop-2 expression via FACS SN-38 95% CI 38 95% CI Median fluorescencePercent IC₅₀ IC₅₀ IC₅₀ IC₅₀ ADC/free SN-38 Cell line (background)positive (nmol/L) (nmol/L) (nmol/L) (nmol/L) ratio Calu-3 282.2 (4.7)99.6% 7.19 5.77-8.95 9.97  8.12-12.25 1.39 COLO 205 141.5 (4.5) 99.5%1.02 0.66-1.57 1.95  1.26-3.01 1.91 Capan-1 100.0 (5.0) 94.2% 3.502.17-5.65 6.99  5.02-9.72 2.00 PC-3  46.2 (5.5) 73.6% 1.86 1.16-2.994.24  2.99-6.01 2.28 SK-MES-1  44.0 (3.5) 91.2% 8.61 6.30-11.76 23.1417.98-29.78 2.69 BxPC-3  26.4 (3.1) 98.3% 1.44 1.04-2.00 4.03  3.25-4.982.80

The MTD of irinotecan (24 mg SN-38/kg, q2d×5) was as effective ashRS7-CL2-SN-38 in COLO 205 cells, because mouse serum can moreefficiently convert irinotecan to SN-38 (Morton et al., 2000, Cancer Res60:4206-10) than human serum, but the SN-38 dose in irinotecan (2,400 μgcumulative) was 37.5-fold greater than with the conjugate (64 μg total).

Animals bearing Capan-1 (FIG. 5C) showed no significant response toirinotecan alone when given at an SN-38-dose equivalent to thehRS7-CL2-SN-38 conjugate (e.g., on day 35, average tumor size was0.04±0.05 cm³ in animals given 0.4 mg SN-38/kg hRS7-SN-38 vs. 1.78±0.62cm³ in irinotecan-treated animals given 0.4 mg/kg SN-38; AUC_(day35)P<0.001; FIG. 5C). When the irinotecan dose was increased 10-fold to 4mg/kg SN-38, the response improved, but still was not as significant asthe conjugate at the 0.4 mg/kg SN-38 dose level (TV=0.17±0.18 cm³ vs.1.69±0.47 cm³, AUC_(day49)P<0.001) (FIG. 5C). An equal dose ofnontargeting hA20-CL2-SN-38 also had a significant antitumor effect ascompared to irinotecan-treated animals, but the specific hRS7 conjugatewas significantly better than the irrelevant ADC (TV=0.17±0.18 cm³ vs.0.80±0.68 cm³, AUC_(day49)P<0.018) (FIG. 5C).

Studies with the hRS7-CL2A-SN-38 ADC were then extended to 2 othermodels of human epithelial cancers. In mice bearing BxPC-3 humanpancreatic tumors (FIG. 5D), hRS7-CL2A-SN-38 again significantlyinhibited tumor growth in comparison to control mice treated with salineor an equivalent amount of nontargeting hA20-CL2A-SN-38 (TV=0.24±0.11cm³ vs. 1.17±0.45 cm³ and 1.05±0.73 cm³, respectively;AUC_(day21)P<0.001), or irinotecan given at a 10-fold higher SN-38equivalent dose (TV=0.27±0.18 cm³ vs. 0.90±0.62 cm³, respectively;AUC_(day25)P<0.004) (FIG. 5D). Interestingly, in mice bearing SK-MES-1human squamous cell lung tumors treated with 0.4 mg/kg of the ADC (FIG.5E), tumor growth inhibition was superior to saline or unconjugated hRS7IgG (TV=0.36±0.25 cm³ vs. 1.02±0.70 cm³ and 1.30±1.08 cm³, respectively;AUC_(28 days), P<0.043), but nontargeting hA20-CL2A-SN-38 or the MTD ofirinotecan provided the same antitumor effects as the specifichRS7-SN-38 conjugate (FIG. 5E). In all murine studies, the hRS7-SN-38ADC was well tolerated in terms of body weight loss (not shown).

Biodistribution of hRS7-CL2A-SN-38—The biodistributions ofhRS7-CL2A-SN-38 or unconjugated hRS7 IgG were compared in mice bearingSK-MES-1 human squamous cell lung carcinoma xenografts (not shown),using the respective ¹¹¹In-labeled substrates. A pharmacokineticanalysis was performed to determine the clearance of hRS7-CL2A-SN-38relative to unconjugated hRS7 (not shown). The ADC cleared faster thanthe equivalent amount of unconjugated hRS7, with the ADC exhibiting ˜40%shorter half-life and mean residence time. Nonetheless, this had aminimal impact on tumor uptake (not shown). Although there weresignificant differences at the 24- and 48-hour timepoints, by 72 hours(peak uptake) the amounts of both agents in the tumor were similar.Among the normal tissues, hepatic and splenic differences were the moststriking (not shown). At 24 hours postinjection, there was >2-fold morehRS7-CL2A-SN-38 in the liver than hRS7 IgG (not shown). Conversely, inthe spleen there was 3-fold more parental hRS7 IgG present at peakuptake (48-hour timepoint) than hRS7-CL2A-SN-38 (not shown). Uptake andclearance in the rest of the tissues generally reflected differences inthe blood concentration (not shown).

Because twice-weekly doses were given for therapy, tumor uptake in agroup of animals that first received a predose of 0.2 mg/kg (250 μgprotein) of the hRS7 ADC 3 days before the injection of the¹¹¹In-labeled antibody was examined. Tumor uptake of¹¹¹In-hRS7-CL2A-SN-38 in predosed mice was substantially reduced atevery timepoint in comparison to animals that did not receive thepredose (e.g., at 72 hours, predosed tumor uptake was 12.5%±3.8% ID/gvs. 25.4%±8.1% ID/g in animals not given the predose; P=0.0123; notshown). Predosing had no appreciable impact on blood clearance or tissueuptake (not shown). These studies suggest that in some tumor models,tumor accretion of the specific antibody can be reduced by the precedingdose(s), which likely explains why the specificity of a therapeuticresponse could be diminished with increasing ADC doses and why furtherdose escalation is not indicated.

Tolerability of hRS7-CL2A-SN-38 in Swiss-Webster Mice and CynomolgusMonkeys

Swiss-Webster mice tolerated 2 doses over 3 days, each of 4, 8, and 12mg SN-38/kg of the hRS7-CL2A-SN-38, with minimal transient weight loss(not shown). No hematopoietic toxicity occurred and serum chemistriesonly revealed elevated aspartate transaminase (AST, FIG. 6A) and alaninetransaminase (ALT, FIG. 6B). Seven days after treatment, AST rose abovenormal levels (>298 U/L) in all 3 treatment groups (FIG. 6A), with thelargest proportion of mice being in the 2×8 mg/kg group. However, by 15days posttreatment, most animals were within the normal range. ALTlevels were also above the normal range (>77 U/L) within 7 days oftreatment (FIG. 6B) and with evidence of normalization by Day 15. Liversfrom all these mice did not show histologic evidence of tissue damage(not shown). In terms of renal function, only glucose and chloridelevels were somewhat elevated in the treated groups. At 2×8 mg/kg, 5 of7 mice had slightly elevated glucose levels (range of 273-320 mg/dL,upper end of normal 263 mg/dL) that returned to normal by 15 dayspostinjection. Similarly, chloride levels were slightly elevated,ranging from 116 to 127 mmol/L (upper end of normal range 115 mmol/L) inthe 2 highest dosage groups (57% in the 2×8 mg/kg group and 100% of themice in the 2×12 mg/kg group), and remained elevated out to 15 dayspostinjection. This also could be indicative of gastrointestinaltoxicity, because most chloride is obtained through absorption by thegut; however, at termination, there was no histologic evidence of tissuedamage in any organ system examined (not shown).

Because mice do not express Trop-2 identified by hRS7, a more suitablemodel was required to determine the potential of the hRS7 conjugate forclinical use. Immunohistology studies revealed binding in multipletissues in both humans and Cynomolgus monkeys (breast, eye,gastrointestinal tract, kidney, lung, ovary, fallopian tube, pancreas,parathyroid, prostate, salivary gland, skin, thymus, thyroid, tonsil,ureter, urinary bladder, and uterus; not shown). Based on thiscross-reactivity, a tolerability study was performed in monkeys.

The group receiving 2×0.96 mg SN-38/kg of hRS7-CL2A-SN-38 had nosignificant clinical events following the infusion and through thetermination of the study. Weight loss did not exceed 7.3% and returnedto acclimation weights by day 15. Transient decreases were noted in mostof the blood count data (neutrophil and platelet data shown in FIG. 6Cand FIG. 6D), but values did not fall below normal ranges. No abnormalvalues were found in the serum chemistries. Histopathology of theanimals necropsied on day 11 (8 days after last injection) showedmicroscopic changes in hematopoietic organs (thymus, mandibular andmesenteric lymph nodes, spleen, and bone marrow), gastrointestinalorgans (stomach, duodenum, jejunum, ileum, cecum, colon, and rectum),female reproductive organs (ovary, uterus, and vagina), and at theinjection site. These changes ranged from minimal to moderate and werefully reversed at the end of the recovery period (day 32) in alltissues, except in the thymus and gastrointestinal tract, which weretrending towards full recovery at this later timepoint (not shown).

At the 2×1.92 mg SN-38/kg dose level of the conjugate, there was 1 deatharising from gastrointestinal complications and bone marrow suppression,and other animals within this group showed similar, but more severeadverse events than the 2×0.96 mg/kg group (not shown). These dataindicate that dose-limiting toxicities were identical to that ofirinotecan; namely, intestinal and hematologic. Thus, the MTD forhRS7-CL2A-SN-38 lies between 2×0.96 and 1.92 mg SN-38/kg, whichrepresents a human equivalent dose of 2×0.3 to 0.6 mg/kg SN-38.

Discussion

Trop-2 is a protein expressed on many epithelial tumors, including lung,breast, colorectal, pancreas, prostate, and ovarian cancers, making it apotentially important target for delivering cytotoxic agents (Ohmachi etal., 2006, Clin Cancer Res 12:3057-63; Fong et al., 2008, Br J Cancer99:1290-95; Cubas et al., 2009, Biochim Biophys Acta 1796:309-14). TheRS7 antibody internalizes when bound to Trop-2 (Shih et al., 1995,Cancer Res 55:5857s-63s), which enables direct intracellular delivery ofcytotoxics.

SN-38 is a potent topoisomerase-I inhibitor, with IC₅₀ values in thenanomolar range in several cell lines. It is the active form of theprodrug, irinotecan, that is used for the treatment of colorectalcancer, and which also has activity in lung, breast, and brain cancers.We reasoned that a directly targeted SN-38, in the form of an ADC, wouldbe a significantly improved therapeutic over CPT-11, by overcoming thelatter's low and patient-variable bioconversion to active SN-38(Mathijssen et al., 2001, Clin Cancer Res 7:2182-94).

The Phe-Lys peptide inserted in the original CL2 derivative allowed forpossible cleavage via cathepsin B. To simplify the synthetic process, inCL2A the phenylalanine was eliminated, and thus the cathepsin B cleavagesite was removed. Interestingly, this product had a better-definedchromatographic profile compared to the broad profile obtained with CL2(not shown), but more importantly, this change had no impact on theconjugate's binding or stability, and surprisingly produced a smallincrease in potency in side-by-side testing.

In vitro cytotoxicity of hRS7 ADC against a range of solid tumor celllines consistently had IC₅₀ values in the nmol/L range. However, cellsexposed to free SN-38 demonstrated a lower IC₅₀ value compared to theADC. This disparity between free and conjugated SN-38 was also reportedfor ENZ-2208 (Sapra et al., 2008, Clin Cancer Res 14:1888-96, Zhao etal., 2008, Bioconjug Chem 19:849-59) and NK012 (Koizumi et al., 2006,Cancer Res 66:10048-56). ENZ-2208 utilizes a branched PEG to link about3.5 to 4 molecules of SN-38 per PEG, whereas NK012 is a micellenanoparticle containing 20% SN-38 by weight. With our ADC, thisdisparity (i.e., ratio of potency with free vs. conjugated SN-38)decreased as the Trop-2 expression levels increased in the tumor cells,suggesting an advantage to targeted delivery of the drug. In terms of invitro serum stability, both the CL2- and CL2A-SN-38 forms of hRS7-SN-38yielded a t/_(1/2) of ˜20 hours, which is in contrast to the shortt/_(1/2) of 12.3 minutes reported for ENZ-2208 (Zhao et al., 2008,Bioconjug Chem 19:849-59), but similar to the 57% release of SN-38 fromNK012 under physiological conditions after 24 hours (Koizumi et al.,2006, Cancer Res 66:10048-56). Treatment of tumor-bearing mice withhRS7-SN-38 (either with CL2-SN-38 or CL2A-SN-38) significantly inhibitedtumor growth in 5 different tumor models. In 4 of them, tumorregressions were observed, and in the case of Calu-3, all mice receivingthe highest dose of hRS7-SN-38 were tumor-free at the conclusion ofstudy. Unlike in humans, irinotecan is very efficiently converted toSN-38 by a plasma esterase in mice, with a greater than 50% conversionrate, and yielding higher efficacy in mice than in humans (Morton etal., 2000, Cancer Res 60:4206-10; Furman et al., 1999, J Clin Oncol17:1815-24). When irinotecan was administered at 10-fold higher orequivalent SN-38 levels, hRS7-SN-38 was significantly better incontrolling tumor growth. Only when irinotecan was administered at itsMTD of 24 mg/kg q2dx5 (37.5-fold more SN-38) did it equal theeffectiveness of hRS7-SN-38. In patients, we would expect this advantageto favor hRS7-CL2A-SN-38 even more, because the bioconversion ofirinotecan would be substantially lower.

We also showed in some antigen-expressing cell lines, such as SK-MES-1,that using an antigen-binding ADC does not guarantee better therapeuticresponses than a nonbinding, irrelevant conjugate. This is not anunusual or unexpected finding. Indeed, the nonbinding SN-38 conjugatesmentioned earlier enhance therapeutic activity when compared toirinotecan, and so an irrelevant IgG-SN-38 conjugate is expected to havesome activity. This is related to the fact that tumors have immature,leaky vessels that allow the passage of macromolecules better thannormal tissues (Jain, 1994, Sci Am 271:58-61). With our conjugate, 50%of the SN-38 will be released in ˜13 hours when the pH is lowered to alevel mimicking lysosomal levels (e.g., pH 5.3 at 37° C.; data notshown), whereas at the neutral pH of serum, the release rate is reducednearly 2-fold. If an irrelevant conjugate enters an acidic tumormicroenvironment, it is expected to release some SN-38 locally. Otherfactors, such as tumor physiology and innate sensitivities to the drug,will also play a role in defining this “baseline” activity. However, aspecific conjugate with a longer residence time should have enhancedpotency over this baseline response as long as there is ample antigen tocapture the specific antibody. Biodistribution studies in the SK-IVIES-1model also showed that if tumor antigen becomes saturated as aconsequence of successive dosing, tumor uptake of the specific conjugateis reduced, which yields therapeutic results similar to that found withan irrelevant conjugate.

Although it is challenging to make direct comparisons between our ADCand the published reports of other SN-38 delivery agents, some generalobservations can be made. In our therapy studies, the highest individualdose was 0.4 mg/kg of SN-38. In the Calu-3 model, only 4 injections weregiven for a total cumulative dose of 1.6 mg/kg SN-38 or 32 μg SN-38 in a20 g mouse. Multiple studies with ENZ-2208 were done using its MTD of 10mg/kg×5 (Sapra et al., 2008, Clin Cancer Res 14:1888-96; Pastorini etal., 2010, Clin Cancer Res 16:4809-21), and preclinical studies withNK012 involved its MTD of 30 mg/kg×3 (Koizumi et al., 2006, Cancer Res66:10048-56). Thus, significant antitumor effects were obtained withhRS7-SN-38 at 30-fold and 55-fold less SN-38 equivalents than thereported doses in ENZ-2208 and NK012, respectively. Even with 10-foldless hRS7 ADC (0.04 mg/kg), significant antitumor effects were observed,whereas lower doses of ENZ-2208 were not presented, and when the NK012dose was lowered 4-fold to 7.5 mg/kg, efficacy was lost (Koizumi et al.,2006, Cancer Res 66:10048-56). Normal mice showed no acute toxicity witha cumulative dose over 1 week of 24 mg/kg SN-38 (1,500 mg/kg of theconjugate), indicating that the MTD was higher. Thus, tumor-bearinganimals were effectively treated with 7.5- to 15-fold lower amounts ofSN-38 equivalents.

Biodistribution studies revealed the hRS7-CL2A-SN-38 had similar tumoruptake as the parental hRS7 IgG, but cleared substantially faster with2-fold higher hepatic uptake, which may be due to the hydrophobicity ofSN-38. With the ADC being cleared through the liver, hepatic andgastrointestinal toxicities were expected to be dose limiting. Althoughmice had evidence of increased hepatic transaminases, gastrointestinaltoxicity was mild at best, with only transient loss in weight and noabnormalities noted upon histopathologic examination. Interestingly, nohematological toxicity was noted. However, monkeys showed an identicaltoxicity profile as expected for irinotecan, with gastrointestinal andhematological toxicity being dose-limiting.

Because Trop-2 recognized by hRS7 is not expressed in mice, it wasimportant to perform toxicity studies in monkeys that have a similartissue expression of Trop-2 as humans. Monkeys tolerated 0.96 mg/kg/dose(˜12 mg/m²) with mild and reversible toxicity, which extrapolates to ahuman dose of ˜0.3 mg/kg/dose (˜11 mg/m²). In a Phase I clinical trialof NK012, patients with solid tumors tolerated 28 mg/m² of SN-38 every 3weeks with Grade 4 neutropenia as dose-limiting toxicity (DLT; Hamaguchiet al., 2010, Clin Cancer Res 16:5058-66). Similarly, Phase I clinicaltrials with ENZ-2208 revealed dose-limiting febrile neutropenia, with arecommendation to administer 10 mg/m² every 3 weeks or 16 mg/m² ifpatients were administered G-CSF (Kurzrock et al., AACR-NCI-EORTCInternational Conference on Molecular Targets and Cancer Therapeutics;2009 Nov. 15-19; Boston, Mass.; Poster No C216; Patnaik et al.,AACR-NCI-EORTC International Conference on Molecular Targets and CancerTherapeutics; 2009 Nov. 15-19; Boston, Mass.; Poster No C221). Becausemonkeys tolerated a cumulative human equivalent dose of 22 mg/m², itappears that even though hRS7 binds to a number of normal tissues, theMTD for a single treatment of the hRS7 ADC could be similar to that ofthe other nontargeting SN-38 agents. Indeed, the specificity of theanti-Trop-2 antibody did not appear to play a role in defining the DLT,because the toxicity profile was similar to that of irinotecan. Moreimportantly, if antitumor activity can be achieved in humans as in micethat responded with human equivalent dose of just at 0.03 mg SN-38equivalents/kg/dose, then significant antitumor responses may berealized clinically.

In conclusion, toxicology studies in monkeys, combined with in vivohuman cancer xenograft models in mice, have indicated that this ADCtargeting Trop-2 is an effective therapeutic in several tumors ofdifferent epithelial origin.

Example 4. Cell Binding Assay of Anti-Trop-2 Antibodies

Two different murine monoclonal antibodies against human Trop-2 wereobtained for ADC conjugation. The first, 162-46.2, was purified from ahybridoma (ATCC, HB-187) grown up in roller-bottles. A second antibody,MAB650, was purchased from R&D Systems (Minneapolis, Minn.). For acomparison of binding, the Trop-2 positive human gastric carcinoma,NCI-N87, was used as the target. Cells (1.5×10⁵/well) were plated into96-well plates the day before the binding assay. The following morning,a dose/response curve was generated with 162-46.2, MAB650, and murineRS7 (0.03 to 66 nM). These primary antibodies were incubated with thecells for 1.5 h at 4° C. Wells were washed and an anti-mouse-HRPsecondary antibody was added to all the wells for 1 h at 4° C. Wells arewashed again followed by the addition of a luminescence substrate.Plates were read using Envision plate reader and values are reported asrelative luminescent units.

All three antibodies had similar KD-values of 0.57 nM for RS7, 0.52 nMfor 162-46.2 and 0.49 nM for MAB650. However, when comparing the maximumbinding (Borax) of 162-46.2 and MAB650 to RS7 they were reduced by 25%and 50%, respectively (BMax 11,250 for RS7, 8,471 for 162-46.2 and 6,018for MAB650) indicating different binding properties in comparison toRS7.

Example 5. Cytotoxicity of Anti-Trop-2 ADC (MAB650-SN-38)

A novel anti-Trop-2 ADC was made with SN-38 and MAB650, yielding a meandrug to antibody substitution ratio of 6.89. Cytotoxicity assays wereperformed to compare the MAB650-SN-38 and hRS7-SN-38 ADCs using twodifferent human pancreatic adenocarcinoma cell lines (BxPC-3 andCapan-1) and a human triple negative breast carcinoma cell line(MDA-MB-468) as targets.

One day prior to adding the ADCs, cells were harvested from tissueculture and plated into 96-well plates. The next day cells were exposedto hRS7-SN-38, MAB650-SN-38, and free SN-38 at a drug range of3.84×10⁻¹² to 2.5×10⁻⁷ M. Unconjugated MAB650 was used as a control atprotein equivalent doses as the MAB650-SN-38. Plates were incubated at37° C. for 96 h. After this incubation period, an MTS substrate wasadded to all of the plates and read for color development at half-hourintervals until an OD_(492 nm) of approximately 1.0 was reached for theuntreated cells. Growth inhibition was measured as a percent of growthrelative to untreated cells using Microsoft Excel and Prism software(non-linear regression to generate sigmoidal dose response curves whichyield IC₅₀-values.

As shown in FIG. 7A-B, hRS7-SN-38 and MAB650-SN-38 had similargrowth-inhibitory effects with IC₅₀-values in the low nM range which istypical for SN-38-ADCs in these cell lines. In the human Capan-1pancreatic adenocarcinoma cell line (FIG. 7A), the hRS7-SN-38 ADC showedan IC₅₀ of 3.5 nM, compared to 4.1 nM for the MAB650-SN-38 ADC and 1.0nM for free SN-38. In the human BxPC-3 pancreatic adenocarcinoma cellline (FIG. 7B), the hRS7-SN-38 ADC showed an IC₅₀ of 2.6 nM, compared to3.0 nM for the MAB650-SN-38 ADC and 1.0 nM for free SN-38. In the humanNCI-N87 gastric adenocarcinoma cell line (FIG. 7C), the hRS7-SN-38 ADCshowed an IC₅₀ of 3.6 nM, compared to 4.1 nM for the MAB650-SN-38 ADCand 4.3 nM for free SN-38.

In summary, in these in vitro assays, the SN-38 conjugates of twoanti-Trop-2 antibodies, hRS7 and MAB650, showed equal efficacies againstseveral tumor cell lines, which was similar to that of free SN-38.Because the targeting function of the anti-Trop-2 antibodies would be amuch more significant factor in vivo than in vitro, the data supportthat anti-Trop-2-SN-38 ADCs as a class would be highly efficacious invivo, as demonstrated in the Examples above for hRS7-SN-3 8.

Example 6. Cytotoxicity of Anti-Trop-2 ADC (162-46.2-SN-38)

A novel anti-Trop-2 ADC was made with SN-38 and 162-46.2, yielding adrug to antibody substitution ratio of 6.14. Cytotoxicity assays wereperformed to compare the 162-46.2-SN-38 and hRS7-SN-38 ADCs using twodifferent Trop-2-positive cell lines as targets, the BxPC-3 humanpancreatic adenocarcinoma and the MDA-MB-468 human triple negativebreast carcinoma.

One day prior to adding the ADC, cells were harvested from tissueculture and plated into 96-well plates at 2000 cells per well. The nextday cells were exposed to hRS7-SN-38, 162-46.2-SN-38, or free SN-38 at adrug range of 3.84×10⁻¹² to 2.5×10⁻⁷ M. Unconjugated 162-46.2 and hRS7were used as controls at the same protein equivalent doses as the162-46.2-SN-38 and hRS7-SN-38, respectively. Plates were incubated at37° C. for 96 h. After this incubation period, an MTS substrate wasadded to all of the plates and read for color development at half-hourintervals until untreated control wells had an OD_(492mn) reading ofapproximately 1.0. Growth inhibition was measured as a percent of growthrelative to untreated cells using Microsoft Excel and Prism software(non-linear regression to generate sigmoidal dose response curves whichyield IC₅₀-values).

As shown in FIG. 8A and FIG. 8B, the 162-46.2-SN-38 ADC had a similarIC₅₀-values when compared to hRS7-SN-38. When tested against the BxPC-3human pancreatic adenocarcinoma cell line (FIG. 8A), hRS7-SN-38 had anIC₅₀ of 5.8 nM, compared to 10.6 nM for 162-46.2-SN-38 and 1.6 nM forfree SN-38. When tested against the MDA-MB-468 human breastadenocarcinoma cell line (FIG. 8B), hRS7-SN-38 had an IC₅₀ of 3.9 nM,compared to 6.1 nM for 162-46.2-SN-38 and 0.8 nM for free SN-38. Thefree antibodies alone showed little cytotoxicity to either Trop-2positive cancer cell line.

In summary, comparing the efficacies in vitro of three differentanti-Trop-2 antibodies conjugated to the same cytotoxic drug, all threeADCs exhibited equivalent cytotoxic effects against a variety of Trop-2positive cancer cell lines. These data support that the class ofanti-Trop-2 antibodies, incorporated into drug-conjugated ADCs, areeffective anti-cancer therapeutic agents for Trop-2 expressing solidtumors.

Example 7. Clinical Trials with IMMU-132 Anti-Trop-2 ADC Comprising hRS7Antibody Conjugated to SN-38 Summary

The present Example reports results from a phase I clinical trial andongoing phase II extension with IMMU-132, an ADC of the internalizing,humanized, hRS7 anti-Trop-2 antibody conjugated by a pH-sensitive linkerto SN-38 (mean drug-antibody ratio=7.6). Trop-2 is a type Itransmembrane, calcium-transducing, protein expressed at high density(˜1×10⁵), frequency, and specificity by many human carcinomas, withlimited normal tissue expression. Preclinical studies in nude micebearing Capan-1 human pancreatic tumor xenografts have revealed IMMU-132is capable of delivering as much as 120-fold more SN-38 to tumor thanderived from a maximally tolerated irinotecan therapy.

The present Example reports the initial Phase I trial of 25 patients whohad failed multiple prior therapies (some including topoisomerase-I/IIinhibiting drugs), and the ongoing Phase II extension now reporting on69 patients, including in colorectal (CRC), small-cell and non-smallcell lung (SCLC, NSCLC, respectively), triple-negative breast (TNBC),pancreatic (PDC), esophageal, and other cancers.

As discussed in detail below, Trop-2 was not detected in serum, but wasstrongly expressed (≥2⁺ immunohistochemical staining) in most archivedtumors. In a 3+3 trial design, IMMU-132 was given on days 1 and 8 inrepeated 21-day cycles, starting at 8 mg/kg/dose, then 12 and 18 mg/kgbefore dose-limiting neutropenia. To optimize cumulative treatment withminimal delays, phase II is focusing on 8 and 10 mg/kg (n=30 and 14,respectively). In 49 patients reporting related AE at this time,neutropenia ≥Grade 3 occurred in 28% (4% Grade 4). Most commonnon-hematological toxicities initially in these patients have beenfatigue (55%; ≥G3=9%), nausea (53%; ≥G3=0%), diarrhea (47%; ≥G3=9%),alopecia (40%), and vomiting (32%; ≥G3=2%); alopecia also occurredfrequently. Homozygous UGT1A1 *28/*28 was found in 6 patients, 2 of whomhad more severe hematological and GI toxicities.

In the Phase I and the expansion phases, there are now 48 patients(excluding PDC) who are assessable by RECIST/CT for best response. Seven(15%) of the patients had a partial response (PR), including patientswith CRC (N=1), TNBC (N=2), SCLC (N=2), NSCLC (N =1), and esophagealcancers (N=1), and another 27 patients (56%) had stable disease (SD),for a total of 38 patients (79%) with disease response; 8 of 13CT-assessable PDC patients (62%) had SD, with a median time toprogression (TTP) of 12.7 wks compared to 8.0 weeks in their last priortherapy. The TTP for the remaining 48 patients is 12.6+ wks (range 6.0to 51.4 wks). Plasma CEA and CA19-9 correlated with responses who hadelevated titers of these antigens in their blood. No anti-hRS7 oranti-SN-38 antibodies were detected despite dosing over months.

The conjugate cleared from the serum within 3 days, consistent with invivo animal studies where 50% of the SN-38 was released daily, with >95%of the SN-38 in the serum being bound to the IgG in a non-glucoronidatedform, and at concentrations as much as 100-fold higher than SN-38reported in patients given irinotecan. These results show that thehRS7-SN-38-containing ADC is therapeutically active in metastatic solidcancers, with manageable diarrhea and neutropenia.

Pharmacokinetics

Two ELISA methods were used to measure the clearance of the IgG (capturewith anti-hRS7 idiotype antibody) and the intact conjugate (capture withanti-SN-38 IgG/probe with anti-hRS7 idiotype antibody). SN-38 wasmeasured by HPLC. Total IMMU-132 fraction (intact conjugate) clearedmore quickly than the IgG (not shown), reflecting known gradual releaseof SN-38 from the conjugate. HPLC determination of SN-38 (Unbound andTOTAL) showed >95% the SN-38 in the serum was bound to the IgG. Lowconcentrations of SN-38G suggest SN-38 bound to the IgG is protectedfrom glucoronidation. Comparison of ELISA for conjugate and SN-38 HPLCrevealed both overlap, suggesting the ELISA is a surrogate formonitoring SN-38 clearance.

A summary of the dosing regiment and patient pool is provided in Table7.

TABLE 7 Clinical Trial Parameters Dosing regimen Once weekly for 2 weeksadministered every 21 days for up to 8 cycles. In the initialenrollment, the planned dose was delayed and reduced if ≥ Grade 2treatment-related toxicity; protocol was amended to dose delay andreduction only in the event of ≥ Grade 3 toxicity. Dose level cohorts 8,12, 18 mg/kg; later reduced to an intermediate dose level of 10 mg/kg.Cohort size Standard Phase I [3 + 3] design; expansion includes ~15patients in select cancers. DLT Grade 4 ANC ≥ 7 d; ≥Grade 3 febrileneutropenia of any duration; G4 Plt ≥ 5 d; G4 Hgb; Grade 4 N/V/D anyduration/G3 N/V/D for >48 h; G3 infusion-related reactions; related ≥G3non-hematological toxicity. Maximum Maximum dose where ≥2/6 patientstolerate 1^(st) 21-d cycle w/o delay or Acceptable Dose reduction or ≥G3 toxicity. (MAD) Patients Metastatic colorectal, pancreas, gastric,esophageal, lung (NSCLC, SCLC), triple-negative breast (TNBC), prostate,ovarian, renal, urinary bladder, head/neck, hepatocellular.Refractory/relapsed after standard treatment regimens for metastaticcancer. Prior irinotecan-containing therapy NOT required for enrollment.No bulky lesion >5 cm. Must be 4 weeks beyond any major surgery, and 2weeks beyond radiation or chemotherapy regimen. Gilbert's disease orknown CNS metastatic disease are excluded.

Clinical Trial Status

A total of 69 patients (including 25 patients in Phase I) with diversemetastatic cancers having a median of 3 prior therapies were reported.Eight patients had clinical progression and withdrew before CTassessment. Thirteen CT-assessable pancreatic cancer patients wereseparately reported. The median TTP (time to progression) in PDCpatients was 11.9 wks (range 2 to 21.4 wks) compared to median 8 wks TTPfor the preceding last therapy.

A total of 48 patients with diverse cancers had at least 1 CT-assessmentfrom which Best Response (FIG. 9) and Time to Progression (TTP; FIG. 20)were determined. To summarize the Best Response data, of 8 assessablepatients with TNBC (triple-negative breast cancer), there were 2 PR(partial response), 4 SD (stable disease) and 2 PD (progressive disease)for a total response [PR+SD] of 6/8 (75%). For SCLC (small cell lungcancer), of 4 assessable patients there were 2 PR, 0 SD and 2 PD for atotal response of 2/4 (50%). For CRC (colorectal cancer), of 18assessable patients there were 1 PR, 11 SD and 6 PD for a total responseof 12/18 (67%). For esophageal cancer, of 4 assessable patients therewere 1 PR, 2 SD and 1 PD for a total response of 3/4 (75%). For NSCLC(non-small cell lung cancer), of 5 assessable patients there were 1 PR,3 SD and 1 PD for a total response of 4/5 (80%). Over all patientstreated, of 48 assessable patients there were 7 PR, 27 SD and 14 PD fora total response of 34/48 (71%). These results demonstrate that theanti-TROP-2 ADC (hRS7-SN-38) showed significant clinical efficacyagainst a wide range of solid tumors in human patients.

The reported side effects of therapy (adverse events) are summarized inTable 8. As apparent from the data of Table 8, the therapeutic efficacyof hRS7-SN-38 was achieved at dosages of ADC showing an acceptably lowlevel of adverse side effects.

TABLE 8 Related Adverse Events Listing for IMMU-132-01 Criteria: Total ≥10% or ≥Grade 3 N = 47 patients TOTAL Grade 3 Grade 4 Fatigue 55% 4 (9%)0 Nausea 53% 0 0 Diarrhea 47% 4 (9%) 0 Neutropenia 43% 11 (24%) 2 (4%)Alopecia 40% — — Vomiting 32% 1 (2%) 0 Anemia 13% 2 (4%) 0 Dysgeusia 15%0 0 Pyrexia 13% 0 0 Abdomina pain 11% 0 0 Hypokalemia 11% 1 (2%) 0 WBCDecrease  6% 1 (2%) 0 Febrile Neutropenia  6% 1 (2%) 2 (4%) Deep veinthrombosis  2% 1 (2%) 0 Grading by CTCAE v 4.0

The study reported in Table 8 has continued, with 261 patients enrolledto date. The results (not shown) have generally followed along the linesindicated in Table 8, with only neutropenia showing an incidence ofGrade 3 or higher adverse events of over 10% of the patients tested. Forall other adverse events, the incidence of Grade 3 or higher responseswas less than 10%. This distinguishes the instant immunoconjugates fromthe great majority of ADCs and in certain embodiments, the claimedmethods and compositions relate to anti-Trop-2 ADCs that show efficacyin diverse solid tumors, with an incidence of Grade 3 or higher adverseevents of less than 10% of patients for all adverse events other thanneutropenia. In a follow-up study, in a total of 421 samples from 121patients with baseline and at least one follow-up sample available, noanti-hRS7 or anti-SN-38 antibody response has been detected, despiterepeated cycles of treatment.

Exemplary partial responses to the anti-Trop-2 ADC were confirmed by CTdata (not shown). As an exemplary PR in CRC, a 62 year-old woman firstdiagnosed with CRC underwent a primary hemicolectomy. Four months later,she had a hepatic resection for liver metastases and received 7 mos oftreatment with FOLFOX and 1 mo 5FU. She presented with multiple lesionsprimarily in the liver (3+Trop-2 by immunohistology), entering thehRS7-SN-38 trial at a starting dose of 8 mg/kg about 1 year afterinitial diagnosis. On her first CT assessment, a PR was achieved, with a37% reduction in target lesions (not shown). The patient continuedtreatment, achieving a maximum reduction of 65% decrease after 10 monthsof treatment (not shown) with decrease in CEA from 781 ng/mL to 26.5ng/mL), before progressing 3 months later.

As an exemplary PR in NSCLC, a 65 year-old male was diagnosed with stageIIIB NSCLC (sq. cell). Initial treatment of caboplatin/etoposide (3 mo)in concert with 7000 cGy XRT resulted in a response lasting 10 mo. Hewas then started on Tarceva maintenance therapy, which he continueduntil he was considered for IMMU-132 trial, in addition to undergoing alumbar laminectomy. He received first dose of IMMU-132 after 5 months ofTarceva, presenting at the time with a 5.6 cm lesion in the right lungwith abundant pleural effusion. He had just completed his 6^(th) dosetwo months later when the first CT showed the primary target lesionreduced to 3.2 cm (not shown).

As an exemplary PR in SCLC, a 65 year-old woman was diagnosed withpoorly differentiated SCLC. After receiving carboplatin/etoposide(Topoisomerase-II inhibitor) that ended after 2 months with no response,followed with topotecan (Topoisomerase-I inhibitor) that ended after 2months, also with no response, she received local XRT (3000 cGy) thatended 1 month later. However, by the following month progression hadcontinued. The patient started with IMMU-132 the next month (12 mg/kg;reduced to 6.8 mg/kg; Trop-2 expression 3+), and after two months ofIMMU-132, a 38% reduction in target lesions, including a substantialreduction in the main lung lesion occurred (not shown). The patientprogressed 3 months later after receiving 12 doses.

These results are significant in that they demonstrate that theanti-Trop-2 ADC was efficacious, even in patients who had failed orprogressed after multiple previous therapies. In conclusion, at thedosages used, the primary toxicity was a manageable neutropenia, withfew Grade 3 toxicities. IMMU-132 showed evidence of activity (PR anddurable SD) in relapsed/refractory patients with triple-negative breastcancer, small cell lung cancer, non-small cell lung cancer, colorectalcancer and esophageal cancer, including patients with a previous historyof relapsing on topoisomerase-I inhibitor therapy. These results showefficacy of the anti-Trop-2 ADC in a wide range of cancers that areresistant to existing therapies.

Example 8. Comparative Efficacy of Different Anti-Trop-2 ADCs

The therapeutic efficacy of a murine anti-Trop-2 monoclonal antibody(162-46.2) conjugated with SN-38 was compared to hRS7-SN-38antibody-drug conjugate (ADC) in mice bearing human gastric carcinomaxenografts (NCI-N87). NCI-N87 cells were expanded in tissue culture andharvested with trypsin/EDTA. Female athymic nude mice were injected s.c.with 200 μL of NCI-N87 cell suspension mixed 1:1 with matrigel such that1×10⁷ cells was administered to each mouse. Once tumors reachedapproximately 0.25 cm³ in size (6 days later), the animals were dividedup into seven different treatment groups of nine mice each. For theSN-38 ADCs, mice received 500 μg i.v. injections once a week for twoweeks. Control mice received the non-tumor targeting hA20-SN-38 ADC atthe same dose/schedule. A final group of mice received only saline andserved as the untreated control. Tumors were measured and mice weighedtwice a week. Mice were euthanized for disease progression if theirtumor volumes exceeded 1.0 cm³ in size.

Mean tumor volumes for the SN-38-ADC treated mice are shown in FIG. 11.As determined by area under the curve (AUC), both hRS7-SN-38 and162-46.2-SN-38 significantly inhibited tumor growth when compared tosaline and hA20-SN-38 control mice (P<0.001). Treatment with hRS7-SN-38achieved stable disease in 7 of 9 mice with mean time to tumorprogression (TTP) of 18.4±3.3 days. Mice treated with 162-46.2-SN-38achieved a positive response in 6 of 9 mice with the remaining 3achieving stable disease. Mean TTP was 24.2±6.0 days, which issignificantly longer than hRS7-SN-38 treated animals (P=0.0382). Theseresults confirm the in vivo efficacy of different anti-Trop-2 ADCs fortreatment of human gastric carcinoma.

Example 9. Treatment of Patients with Advanced, Metastatic PancreaticCancer with Anti-Trop-2 ADC Summary

IMMU-132 (hRS7-SN-38) is an anti-Trop-2 ADC comprising the cancer cellinternalizing, humanized, anti-Trop-2 hRS7 antibody, conjugated by apH-sensitive linker to SN-38, the active metabolite of irinotecan, at amean drug-antibody ratio of 7.6. Trop-2 is a type-I transmembrane,calcium-transducing protein expressed at high density, frequency, andspecificity in many epithelial cancers, including pancreatic ductaladenocarcinoma, with limited normal tissue expression. All 29 pancreatictumor microarray specimens tested were Trop-2-positive byimmunohistochemistry, and human pancreatic cancer cell lines were foundto express 115k-891k Trop-2 copies on the cell membrane.

We reported above the results from the IMMU-132 Phase I study enrollingpatients with 13 different tumor types using a 3+3 design. The Phase Idose-limiting toxicity was neutropenia. Over 80% of 24 assessablepatients in this study had long-term stable disease, with partialresponses (RECIST) observed in patients with colorectal (CRC),triple-negative breast (TNBC), small-cell and non-small cell lung (SCLC,NSCLC), and esophageal (EAC) cancers. The present Example reports theresults from the IMMU-132 Phase I/II study cohort of patients withmetastatic PDC. Patients with PDC who failed a median of 2 priortherapies (range 1-5) were given IMMU-132 on days 1 and 8 in repeated21-day cycles.

In the subgroup of PDC patients (N=15), 14 received priorgemcitabine-containing regimens. Initial toxicity data from 9 patientsfound neutropenia [3 of 9≥G3, 33%; and 1 case of G4 febrileneutropenia), which resulted in dose delays or dose reductions. Twopatients had Grade 3 diarrhea; no patient had Grade 3-4 nausea orvomiting. Alopecia (Grades 1-2) occurred in 5 of 9 patients. Bestresponse was assessable in 13 of 14 patients, with 8 stable disease for8 to 21.4 wks (median 12.7 wks; 11.9 wks all 14 patients). One patientwho is continuing treatment has not yet had their first CT assessment.Five had progressive disease by RECIST; 1 withdrew after just 1 dose dueto clinical progression and was not assessable. Serum CA19-9 titersdecreased in 3 of the patients with stable disease by 23 to 72%. Despitemultiple administrations, none of the patients developed an antibodyresponse to IMMU-132 or SN-38. Peak and trough serum samples showed thatIMMU-132 cleared more quickly than the IgG, which is expected based onthe known local release of SN-38 within the tumor cell. Concentrationsof SN-38-bound to IgG in peak samples from one patient given 12 mg/kg ofIMMU-132 showed levels of 4000 ng/mL, which is 40-times higher than theSN-38 titers reported in patients given irinotecan therapy.

We conclude that IMMU-132 is active (long-term stable disease) in 62%(8/13) of PDC patients who failed multiple prior therapies, withmanageable neutropenia and little GI toxicity. Advanced PDC patients canbe given repeated treatment cycles (>6) of 8-10 mg/kg IMMU-132 on days 1and 8 of a 21-day cycle, with some dose adjustments or growth factorsupport for neutropenia in subsequent treatment cycles. These resultsagree with the findings in patients with advanced CRC, TNBC, SCLC,NSCLC, EAC who have shown partial responses and long-term stable diseasewith IMMU-132 administration. In summary, monotherapy IMMU-132 is anovel, efficacious treatment regimen for patients with PDC, includingthose with tumors that were previously resistant to other therapeuticregimens for PDC.

Methods and Results

Trop-2 expression—The expression of Trop-2 on the surface of variouscancer cell lines was determined by flow cytometry using QUANTBRITE® PEbeads. The results for number of Trop-2 molecules detected in thedifferent cell lines was: BxPC-3 pancreatic cancer (891,000); NCI-N87gastric cancer (383,000); MDA-MB-468 breast cacner (341,000); SK-MES-1squamous cell lung cancer (27,000); Capan-1 pancreatic cancer (115,000);AGS gastric cancer (78,000) COLO 205 colon cancer (52,000). Trop-2expression was also observed in 29 of 29 (100%) tissue microarrays ofpancreatic adenocarcinoma (not shown).

SN-38 accumulation—SN-38 accumulation was determined in nude micebearing Capan-1 human pancreatic cancer xenografts (˜0.06-0.27 g). Micewere injected IV with irinotecan 40 mg/kg (773 μg; Total SN-38equivalents=448 μg). This dose is MTD in mice. Human doseequivalent=3.25 mg/kg or ˜126 mg/m². Or mice were injected IV withIMMU-132 1.0 mg (SN-38:antibody ratio=7.6; SN-38 equivalents=20 μg).This dose is well below the MTD in mice. Human equivalent dose ˜4 mg/kgIMMU-132 (˜80 pg/kg SN-38 equivalents). Necropsies were performed on 3animals per interval, in irinotecan injected mice at 5 min, 1, 2, 6 and24 hours or in IMMU-132 injected mice at 1, 6, 24, 48 and 72 h. Tissueswere extracted and analyzed by reversed-phase HPLC analysis for SN-38,SN-38G, and irinotecan. Extracts from IMMU-132-treated animals also wereacid hydrolyzed to release SN-38 from the conjugate (i.e., SN-38(TOTAL]). The results, shown in FIG. 12, demonstrate that the IMMU-132ADC has the potential to deliver 120 times more SN-38 to the tumorcompared to irinotecan, even though 22-fold less SN-38 equivalents wereadministered with the ADC.

IMMU-132 clinical protocol—The protocol used in the phase I/II study wasas indicated in Table 9 below.

TABLE 9 Clinical Protocol Using IMMU-132: OVERVIEW Dosing Once weeklyfor 2 weeks administered every 21 days for up to 8 cycles. regimenPatients with objective responses are allowed to continue beyond 8cycles. In the initial enrollment, the planned dose was delayed andreduced if ≥ Grade 2 treatment-related toxicity; protocol was amendedlater in study to dose delay and reduction only in the event of ≥ Grade3 toxicity. The development of severe toxicities due to treatmentrequires dose reduction by 25% of the assigned dose for 1^(st)occurrence, 50% for 2^(nd) occurrence, and treatment discontinuedentirely in the event of a 3^(rd) occurrence. Dose level 8, 12, 18mg/kg; later reduced to an intermediate dose level of 10 mg/kg. cohortsCohort size Standard Phase I [3 + 3] design; expansion includes 15patients in select cancers. DLT Grade 4 ANC ≥ 7 d; ≥Grade 3 febrileneutropenia of any duration; Grade 4 Platelets ≥5 d; Grade 4 Hgb; Grade4 N/V/D of any duration or any Grade 3 N/V/D for >48 h; Grade 3infusion-related reactions; ≥Grade 3 non- heme toxicity at leastpossibly due to study drug. Maximum Maximum dose where ≥2/6 patientstolerate the full 21-d treatment cycle Acceptable without dose delay orreduction or ≥ Grade 3 toxicity. Dose (MAD) Patients Metastaticcolorectal, pancreas, gastric, esophageal, lung (NSCLC, SCLC),triple-negative breast, prostate, ovarian, renal, urinary bladder, headand neck, hepatocellular. Refractory/relapsed after standard treatmentregimens for metastatic cancer. Prior irinotecan-containing therapy NOTrequired for enrollment. No bulky lesion >5 cm. Must be 4 weeks beyondany major surgery, and 2 weeks beyond radiation or chemotherapy regimen.Gilbert's disease or known CNS metastatic disease are excluded.

Patients were administered IMMU-132 according to the protocol summarizedabove. The response assessment to last prior therapy before IMMU-132treatment is summarized in FIG. 13. The response assessment to IMMU-132administration is shown in FIG. 14. A summary of time to progression(TTP) results following administration of IMMU-132 is shown in FIG. 15.An exemplary case study is as follows. A 34 y/o white male initiallydiagnosed with metastatic pancreatic cancer (liver) had progressed onmultiple chemotherapy regimens, including gemcitabine/Erlotinib/FG-3019,FOLFIRINOX and GTX prior to introduction of IMMU-132 (8 mg/kg dose givendays 1 and 8 of a 21 day cycle). The patient received the drug for 4 mowith good symptomatic tolerance, an improvement in pain, a 72% maximumdecline in CA19-9 (from 15885 U/mL to 4418 U/mL) and stable disease byCT RECIST criteria along with evidence of tumor necrosis. Therapy had tobe suspended due to a liver abscess; the patient expired ˜6 weeks later,6 mo following therapy initiation.

CONCLUSIONS

Preclinical studies indicated that IMMU-132 delivers 120-times theamount of SN-38 to a human pancreatic tumor xenograft than whenirinotecan is given. As part of a larger study enrolling patients withdiverse metastatic solid cancers, the Phase 2 dose of IMMU-132 wasdetermined to be 8 to 10 mg/kg, based on manageable neutropenia anddiarrhea as the major side effects. No anti-antibody or anti-SN-38antibodies have been detected to-date, even with repeated therapeuticcycles.

A study of 14 advanced PDC patients who relapsed after a median of 2prior therapies showed CT-confirmed antitumor activity consisting of8/13 (62%) with stable disease. Median duration of TTP for 13 CTassessable pts was 12.7 weeks compared to 8.0 weeks estimated from lastprior therapy. This ADC, with a known drug of nanomolar toxicity,conjugated to an antibody targeting Trop-2 prevalent on many epithelialcancers, by a linker affording cleavage at the tumor site, represents anew efficacious strategy in pancreatic cancer therapy with ADCs. Incomparison to the present standard of care for pancreatic cancerpatients, the extension of time to progression in pancreatic cancerpatients, particularly in those resistant to multiple prior therapies,was surprising and could not have been predicted.

Example 10. Combining Antibody-Targeted Radiation (Radioimmunotherapy)and Anti-Trop-2-SN-38 ADC Improves Pancreatic Cancer Therapy

We previously reported effective anti-tumor activity in nude micebearing human pancreatic tumors with ⁹⁰Y-humanized PAM4 IgG (hPAM4;⁹⁰Y-clivatuzumab tetraxetan) that was enhanced when combined withgemcitabine (GEM) (Gold et al., Int J. Cancer 109:618-26, 2004; ClinCancer Res 9:3929S-37S, 2003). These studies led to clinical testing offractionated ⁹⁰Y-hPAM4 IgG combined with GEM that is showing encouragingobjective responses. While GEM is known for its radiosensitizingability, alone it is not a very effective therapeutic agent forpancreatic cancer and its dose is limited by hematologic toxicity, whichis also limiting for ⁹⁰Y-hPAM4 IgG.

As discussed in the Examples above, an anti-Trop-2 ADC composed of hRS7IgG linked to SN-38 shows anti-tumor activity in various solid tumors.This ADC is very well tolerated in mice (e.g., ≥60 mg), yet just 4.0 mg(0.5 mg, twice-weekly×4) is significantly therapeutic. Trop-2 is alsoexpressed in most pancreatic cancers.

The present study examined combinations of ⁹⁰Y-hPAM4 IgG with RS7-SN-38in nude mice bearing 0.35 cm³ subcutaneous xenografts of the humanpancreatic cancer cell line, Capan-1. Mice (n=10) were treated with asingle dose of ⁹⁰Y-hPAM4 IgG alone (130 μCi, i.e., the maximum tolerateddose (MTD) or 75 μCi), with RS7-SN-38 alone (as above), or combinationsof the 2 agents at the two ⁹⁰Y-hPAM4 dose levels, with the first ADCinjection given the same day as the ⁹⁰Y-hPAM4. All treatments weretolerated, with ≤15% loss in body weight. Objective responses occurredin most animals, but they were more robust in both of the combinationgroups as compared to each agent given alone. All animals in the0.13-mCi ⁹⁰Y-hPAM4 IgG+hRS7-SN-38 group achieved a tumor-free statewithin 4 weeks, while other animals continued to have evidence ofpersistent disease. These studies provide the first evidence thatcombined radioimmunotherapy and ADC enhances efficacy at safe doses.

In the ongoing PAM4 clinical trials, a four week clinical treatmentcycle is performed. In week 1, subjects are administered a dose of¹¹¹In-hPAM4, followed at least 2 days later by gemcitabine dose. Inweeks 2, 3 and 4, subjects are administered a ⁹⁰Y-hPAM4 dose, followedat least 2 days later by gemcitabine (200 mg/m²). Escalation started at3×6.5 mCi/m². The maximum tolerated dose in front-line pancreatic cancerpatients was 3×15 mCi/m² (hematologic toxicity is dose-limiting). Of 22CT-assessable patients, the disease control rate (CR+PR+SD) was 68%,with 5 (23%) partial responses and 10 (45%) having stabilization as bestresponse by RECIST criteria.

Preparation of Antibody-Drug Conjugate (ADC)

The SN-38 conjugated hRS7 antibody was prepared as described above andaccording to previously described protocols (Moon et al. J Med Chem2008, 51:6916-6926; Govindan et al., Clin Cancer Res 2009.15:6052-6061). A reactive bifunctional derivative of SN-38 (CL2A-SN-38)was prepared. The formula of CL2A-SN-38 is(maleimido-[x]-Lys-PABOCO-20-O-SN-38, where PAB is p-aminobenzyl and ‘x’contains a short PEG). Following reduction of disulfide bonds in theantibody with TCEP, the CL2A-SN-38 was reacted with reduced antibody togenerate the SN-38 conjugated RS7.

⁹⁰Y-hPAM4 is prepared as previously described (Gold et al., Clin CancerRes 2003, 9:3929S-37S; Gold et al., Int J Cancer 2004, 109:618-26).

Combination RAIT+ADC

The Trop-2 antigen is expressed in most epithelial cancers (lung,breast, prostate, ovarian, colorectal, pancreatic) and hRS7-SN-38conjugates are being examined in various human cancer-mouse xenograftmodels. Initial clinical trials with ⁹⁰Y-hPAM4 IgG plus radiosensitizingamounts of GEM are encouraging, with evidence of tumor shrinkage orstable disease. However, therapy of pancreatic cancer is verychallenging. Therefore, a combination therapy was examined to determinewhether it would induce a better response. Specifically, administrationof hRS7-SN-38 at effective, yet non-toxic doses was combined with RAITwith ⁹⁰Y-hPAM4 IgG.

The results demonstrated that the combination of hRS7-SN-38 with⁹⁰Y-hPAM4 was more effective than either treatment alone, or the sum ofthe individual treatments (not shown). At a dosage of 75 μCi ⁹⁰Y-hPAM4,only 1 of 10 mice was tumor-free after 20 weeks of therapy (not shown),the same as observed with hRS7-SN-38 alone (not shown). However, thecombination of hRS7-SN-38 with ⁹⁰Y-hPAM4 resulted in 4 of 10 mice thatwere tumor-free after 20 weeks (not shown), and the remaining subjectsshowed substantial decrease in tumor volume compared with eithertreatment alone (not shown). At 130 μCi ⁹⁰Y-hPAM4 the difference waseven more striking, with 9 of 10 animals tumor-free in the combinedtherapy group compared to 5 of 10 in the RAIT alone group (not shown).These data demonstrate the synergistic effect of the combination ofhRS7-SN-38 with ⁹⁰Y-hPAM4. RAIT+ADC significantly improved time toprogression and increased the frequency of tumor-free treatment. Thecombination of ADC with hRS7-SN-38 added to the MTD of RAIT with⁹⁰Y-hPAM4 had minimal additional toxicity, indicated by the % weightloss of the animal in response to treatment (not shown).

The effect of different sequential treatments on tumor survivalindicated that the optimal effect is obtained when RAIT is administeredfirst, followed by ADC (not shown). In contrast, when ADC isadministered first followed by RAIT, there is a decrease in theincidence of tumor-free animals (not shown). Neither unconjugated hPAM4nor hRS7 antibodies had anti-tumor activity when given alone (notshown).

Example 11. Use of hRS7-SN-38 (IMMU-132) to Treat Therapy-RefractiveMetastatic Breast Cancer

The patient was a 57-year-old woman with stage IV, triple-negative,breast cancer (ER/PR negative, HER-neu negative), originally diagnosedin 2005. She underwent a lumpectomy of her left breast in 2005, followedby Dose-Dense ACT in adjuvant setting in September 2005. She thenreceived radiation therapy, which was completed in November. Localrecurrence of the disease was identified when the patient palpated alump in the contralateral (right) breast in early 2012, and was thentreated with CMF (cyclophosphamide, methotrexate, 5-fluorouracil)chemotherapy. Her disease recurred in the same year, with metastaticlesions in the skin of the chest wall. She then received acarboplatin+TAXOL® chemotherapy regimen, during which thrombocytopeniaresulted. Her disease progressed and she was started on weeklydoxorubicin, which was continued for 6 doses. The skin disease also wasprogressing. An FDG-PET scan on 09/26/12 showed progression of diseaseon the chest wall and enlarged, solid, axillary nodes. The patient wasgiven oxycodone for pain control.

She was given IXEMPRA® from October 2012 until February 2013 (every 2weeks for 4 months), when the chest wall lesion opened up and bled. Shewas then put on XELODA®, which was not tolerated well due to neuropathyin her hands and feet, as well as constipation. The skin lesions wereprogressive and then she was enrolled in the IMMU-132 trial after givinginformed consent. The patient also had a medical history ofhyperthyroidism and visual disturbances, with high risk of CNS disease(however, brain Mill was negative for CNS disease). At the time ofenrollment to this trial, her cutaneous lesions (target) in the rightbreast measured 4.4 cm and 2.0 cm in the largest diameter. She hadanother non-target lesion in the right breast and one enlarged lymphnode each in the right and left axilla.

The first IMMU-132 infusion (12 mg/kg) was started on Mar. 12, 2013,which was tolerated well. Her second infusion was delayed due to Grade 3absolute neutrophil count (ANC) reduction (0.9) on the scheduled day ofinfusion, one week later. After a week delay and after receivingNEULASTA®, her second IMMU-132 was administered, with a 25% dosereduction at 9 mg/kg. Thereafter she has been receiving IMMU-132 onschedule as per protocol, once weekly for 2 weeks, then one week off.Her first response assessment on May 17, 2013, after 3 therapy cycles,showed a 43% decrease in the sum of the long diameter of the targetlesions, constituting a partial response by RECIST criteria. She iscontinuing treatment at the 9 mg/kg dose level. Her overall health andclinical symptoms improved considerably since she started treatment withIMMU-132.

Example 12. Use of hRS7-SN-38 (IMMU-132) to Treat Refractory,Metastatic, Small-Cell Lung Cancer

This is a 65-year-old woman with a diagnosis of small-cell lung cancer,involving her left lung, mediastinal lymph nodes, and MM evidence of ametastasis to the left parietal brain lobe. Prior chemotherapy includescarboplatin, etoposide, and topotecan, but with no response noted.Radiation therapy also fails to control her disease. She is then givenIMMU-132 at a dose of 18 mg/kg once every three weeks for a total of 5infusions. After the second dose, she experiences hypotension and aGrade 2 neutropenia, which improve before the next infusion. After thefifth infusion, a CT study shows 13% shrinkage of her target left lungmass. MRI of the brain also shows a 10% reduction of this metastasis.She continues her IMMU-132 dosing every 3 weeks for another 3 months,and continues to show objective and subjective improvement of hercondition, with a 25% reduction of the left lung mass and a 21%reduction of the brain metastasis.

Example 13. Therapy of a Gastric Cancer Patient with Stage IV MetastaticDisease with hRS7-SN-38 (IMMU-132)

This patient is a 60-year-old male with a history of smoking and periodsof excessive alcohol intake over a 40-year-period. He experiences weightloss, eating discomfort and pain not relieved by antacids, frequentabdominal pain, lower back pain, and most recently palpable nodes inboth axilla. He seeks medical advice, and after a workup is shown tohave an adenocarcinoma, including some squamous features, at thegastro-esophageal junction, based on biopsy via a gastroscope.Radiological studies (CT and FDG-PET) also reveal metastatic disease inthe right and left axilla, mediastinal region, lumbar spine, and liver(2 tumors in the right lobe and 1 in the left, all measuring between 2and 4 cm in diameter). His gastric tumor is resected and he is then puton a course of chemotherapy with epirubicin, cisplatin, and5-fluorouracil. After 4 months and a rest period of 6 weeks, he isswitched to docetaxel chemotherapy, which also fails to control hisdisease, based on progression confirmed by CT measurements of themetastatic tumors and some general deterioration.

The patient is then given therapy with IMMU-132 (hRS7-SN-38) at a doseof 10 mg/kg infused every-other-week for a total of 6 doses, after whichCT studies are done to assess status of his disease. These infusions aretolerated well, with some mild nausea and diarrhea, controlled withsymptomatic medications. The CT studies reveal that the sum of his indexmetastatic lesions has decreased by 28%, so he continues on this therapyfor another 5 courses. Follow-up CT studies show that the diseaseremains about 35% reduced by RECIST criteria from his baselinemeasurements prior to IMMU-132 therapy, and his general condition alsoappears to have improved, with the patient regaining an optimisticattitude toward his disease being under control.

Example 14. Clinical Trials of IMMU-132 in Diverse Trop-2 PositiveCancers

Abstract

Sacituzumab govitecan (IMMU-132, also known as hRS7-CL2A-SN-38) is anantibody-drug conjugate (ADC) targeting Trop-2, a surface glycoproteinexpressed on many epithelial tumors, for delivery of SN-38, the activemetabolite of irinotecan. Unlike most ADCs that use ultratoxic drugs andstable linkers, IMMU-132 uses a moderately toxic drug with a moderatelystable carbonate bond between SN-38 and the linker. Flow cytometry andimmunohistochemistry disclosed Trop-2 is expressed in a wide range oftumor types, including gastric, pancreatic, triple-negative breast(TNBC), colonic, prostate, and lung. While cell-binding experimentsreveal no significant differences between IMMU-132 and parental hRS7antibody, surface plasmon resonance analysis using a Trop-2 CM5 chipshows a significant binding advantage for IMMU-132 over hRS7. Theconjugate retained binding to the neonatal receptor, but lost greaterthan 60% of the antibody-dependent cell-mediated cytotoxicity activitycompared to hRS7.

Exposure of tumor cells to either free SN-38 or IMMU-132 demonstratedthe same signaling pathways, with pJNK1/2 and p21WAF1/Cip1 up-regulationfollowed by cleavage of caspases 9, 7, and 3, ultimately leading topoly-ADP-ribose polymerase cleavage and double-stranded DNA breaks.Pharmacokinetics of the intact ADC in mice reveals a mean residence time(MRT) of 15.4 h, while the carrier hRS7 antibody cleared at a similarrate as unconjugated antibody (MRT=˜300 h). IMMU-132 treatment of micebearing human gastric cancer xenografts (17.5 mg/kg; twice weekly×4weeks) resulted in significant anti-tumor effects compared to micetreated with a non-specific control. Clinically relevant dosing schemesof IMMU-132 administered either every other week, weekly, or twiceweekly in mice bearing human pancreatic or gastric cancer.

The present Phase I trial evaluated this ADC as a potential therapeuticfor pretreated patients with a variety of metastatic solid cancers. Inparticular embodiments, the therapy is of use to treat patients who hadpreviously been found to be resistant to, or had relapsed from, standardanti-cancer treatments, including but not limited to treatment withirinotecan, the parent compound of SN-38. These results were surprisingand unexpected and could not have been predicted.

Sacituzumab govitecan was administered on days 1 and 8 of 21-day cycles,with cycles repeated until dose-limiting toxicity or progression. Doseescalation followed a standard 3+3 scheme with 4 planned dose levels anddose delay or reduction allowed. Twenty-five patients (52-60 years old,3 median prior chemotherapy regimens) were treated at dose levels of 8(N=7), 10 (N=6), 12 (N=9), and 18 (N=3) mg/kg. Neutropenia wasdose-limiting, with 12 mg/kg the maximum tolerated dose for cycle 1, buttoo toxic with repeated cycles. Lower doses were acceptable for extendedtreatment with no treatment-related grade 4 toxicities and grade 3toxicities limited to fatigue (N=3), neutropenia (N=2), diarrhea (N=1),and leukopenia (N=1). Using CT-based RECIST 1.1 criteria, 3 patientsachieved partial responses (triple-negative breast cancer, small-celllung cancer, colon cancer) and 15 others had stable disease as bestresponse; of these, 12 maintained disease control with continuedtreatment for 16-36 weeks. No pre-selection of patients based on tumorTrop-2 expression was undertaken.

It was concluded that sacituzumab govitecan is a promising ADC conjugatewith acceptable toxicity and encouraging therapeutic activity inpatients with difficult-to-treat cancers. The 8 and 10 mg/kg doses wereselected for Phase II studies.

INTRODUCTION

Two new antibody-drug conjugates (ADCs) incorporating differentultratoxic (picomolar potency) drugs have been approved, leading tofurther development of other ADCs based on similar principles, includinguse of ultratoxic drugs (Younes et al., 2011, Nat Rev Drug Discov11:19-20; Sievers & Senter, 2013, Ann Rev Med 64:15-29; Krop & Winer,2014, Clin Cancer Res 20:15-20). Aternatively, Moon et al. (2008, J MedChem 51:6916-26) and Govindan et al. (2009, Clin Cancer Res 15:6052-61)selected SN-38, a topoisomerase I inhibitor that is the activemetabolite of irinotecan, an approved drug with well-known but complexpharmacology (Mathijssen et al., 2001, Clin Cancer Res 7:2182-94).Several linkers for conjugating SN-38 were evaluated for release fromthe IgG at varying rates, from several hours to days (Moon et al., 2008,J Med Chem 51:6916-26; Govindan et al., 2009, Clin Cancer Res15:6052-61; Cardillo et al., 2011, Clin Cancer Res 17:3157-69). Theoptimal linker that was selected, designated CL2A, exhibiting anintermediate conjugate stability in serum, was attached to the hydroxylgroup on SN-38's lactone ring, thereby protecting this ring from openingto the less toxic carboxylate form while bound to the linker, andcontained a short polyethylene glycol moiety to enhance solubility(Cardillo et al., 2011, Clin Cancer Res 17:3157-69). The active form ofSN-38 was liberated when the carbonate bond between the linker and SN-38was cleaved, which occurred at low pH, such as that found in lysosomes,as well as the tumor microenvironment, or possibly through enzymaticdegradation.

The antibody chosen for this ADC targeted a tumor-associated antigen,Trop-2 (trophoblast cell-surface antigen) (Cardillo et al., 2011, ClinCancer Res 17:3157-69), using the humanized RS7 monoclonal antibody thatwas shown previously to internalize (Stein et al., 1993, Int J Cancer55:938-46. Trop-2 is an important tumor target for an ADC, because it isover-expressed on many epithelial tumors, particularly more aggressivetypes (Ambrogi et al., 2014, PLoS One 9:e96993; Cubas et al., 2009,Biochim Biophys Act 1796:309-14; Trerotola et al., 2013, Oncogene32:222-33). Trop-2 is also present on a number of normal tissues, butpreclinical studies in monkeys that express the antigen observed onlydose-limiting neutropenia and diarrhea with this new ADC, with noevidence of appreciable toxicity to the Trop-2-expressing normal tissues(Cardillo et al., 2011, Clin Cancer Res 17:3157-69). Therefore, withpreclinical data demonstrating activity in several human tumor xenograftmodels and showing a high therapeutic window (Cardillo et al., 2011,Clin Cancer Res 17:3157-69), a Phase I clinical trial was initiated todetermine the maximum tolerated and optimal doses of this novel ADC inheavily-pretreated patients with diverse, relapsed/refractory,metastatic epithelial tumors. This trial was registered atClinicalTrials.gov (NCT01631552).

Materials and Methods

Entry criteria—The primary objective was to determine the safety andtolerability of sacituzumab govitecan (IMMU-132) as a single agent. Thetrial was designed as a standard 3+3 Phase I design, starting at a doseof 8 mg/kg per injection, with dosages given weekly for 2 weeks in a3-week treatment cycle.

Male and non-pregnant, non-lactating females ≥18 years of age wereeligible if they had a diagnosis of one of thirteen different types ofepithelial tumors. Although no pre-selection based on Trop-2 expressionwas required, these tumors are expected to have Trop-2 expressionin >75% of the cases based on immunohistology studies on archivalspecimens. Patients were required to have measurable metastatic disease(no single lesions ≥5 cm) and had relapsed or were refractory to atleast one approved standard chemotherapeutic regimen for thatindication. Other key criteria included adequate (grade ≤1) hematology,liver and renal function, and no known history of anaphylactic reactionsto irinotecan, or grade ≥3 gastrointestinal toxicity to prior irinotecanor other topoisomerase-I treatments. Since patients with such diversediseases were allowed, prior irinotecan therapy was not a prerequisite.Patients with Gilbert's disease or those who had not toleratedpreviously administered irinotecan or with known CNS metastatic diseasewere excluded.

Study design—Baseline evaluations were performed within 4 weeks of thestart of treatment, with regular monitoring of blood counts, serumchemistries, vital signs, and any adverse events. Anti-antibody andanti-SN-38 antibody responses were measured by ELISA, with samples takenat baseline and then prior to the start of every even-numbered treatmentcycle. The first CT examination was obtained 6-8 weeks from the start oftreatment and then continued at 8- to 12-week intervals untilprogression. Additional follow-up was required only to monitor anyongoing treatment-related toxicity. Toxicities were graded using the NCICTCAE version 4.0, and efficacy assessed by RECIST 1.1.

An ELISA to detect Trop-2 in serum was developed that has a sensitivityof 2 ng/mL, but after testing 12 patients and finding no evidence ofcirculating Trop-2, no further screening was performed. Although not aneligibility criterion, specimens of previously archived tumors wererequested for Trop-2 determination by immunohistology, using a goatpolyclonal antibody anti-human Trop-2 (R&D Systems, Minneapolis, Minn.),since the epitope recognized by the ADC's antibody, hRS7, is notpreserved in formalin-fixed, paraffin-embedded sections (Stein et al.,1993, Int J Cancer 55:938-46). Staining was performed as describedbelow.

Therapeutic regimen—Lyophilized sacituzumab govitecan was reconstitutedin saline and infused over 2-3 h (100 mg of antibody contained ˜1.6 mgof SN-38, with a mean drug:antibody ratio [DAR] of 7.6:1). Prior to thestart of each infusion, most patients received acetaminophen,anti-histamines (H1 and H2 blockers), and dexamethasone. Prophylacticuse of antiemetics or anti-diarrheal medications was prohibited. Therapyconsisted of 2 consecutive doses given on days 1 and 8 of a 3-weektreatment cycle, with the intent to allow patients to continue treatmentfor up to 8 cycles (i.e., 16 treatments) unless there was unacceptabletoxicity or progression. Patients showing disease stabilization orresponse after 8 cycles could continue treatments.

Dose-limiting toxicities (DLT) were considered as grade ≥3 febrileneutropenia of any duration, grade 3 thrombocytopenia with significantbleeding or grade 4 thrombocytopenia ≥5 days, any grade 3 nausea,vomiting or diarrhea that persisted for >48 h despite optimal medicalmanagement, or grade 4 (life threatening) nausea, vomiting or diarrheaof any duration, or any other grade ≥3 non-hematologic toxicity at leastpossibly due to study drug, as well as the occurrence of any grade 3infusion-related reactions.

The maximum tolerated dose (MTD) was judged on the patient's toleranceto the first treatment cycle. On a scheduled treatment day, any patientwith grade ≥2 treatment-related toxicity, with the exception ofalopecia, had their treatment delayed in weekly increments for up to 2weeks. Treatment was reinitiated once toxicity had resolved to grade ≤1.The protocol also initially required all subsequent treatment doses tobe reduced (25% if recovered within 1 week, 50% if within 2 weeks), butthis was relaxed later in the trial when the protocol was amended topermit supportive care after the first cycle. However, if toxicity didnot recover within 3 weeks or worsened, treatment was terminated.Importantly, a dose delay with reduction did not constitute a DLT, andtherefore this allowed treatments to continue, but at a lower dose.Therefore, a patient requiring a dose delay/reduction who was able tocontinue treatment was not considered assessable for DLT, and thenreplaced.

Since a DLT event resulted in the termination of all further treatments,a secondary objective was to assess a dose level that could be toleratedover multiple cycles of treatment with minimal dose delays orreductions. This dose level was designated the maximum acceptable dose,and required patients to tolerate a given dose level in the first cyclewithout having a delay or reduction during that cycle and leading up tothe start of the second cycle.

Pharmacokinetics and immunogenicity—Blood samples were taken within ˜30min from the end of the infusion (e.g., peak) and then prior to eachsubsequent injection (e.g., trough). Samples were separated and serafrozen for determination of total IgG and sacituzumab govitecanconcentrations by ELISA. Serum samples from seven patients also wereassayed for SN-38 content, both total (representing SN-38 bound to theIgG and free) and free SN-38 (i.e., unbound SN-38).

Results

Patient characteristics—Twenty-five patients were enrolled (Table 10).The median age ranged from 52 to 60 years, with 76% having an ECOG 1performance status, the remaining ECOG 0. Most patients had metastaticpancreatic cancer (PDC) (N=7), followed by triple-negative breast cancer(TNBC) (N=4), colorectal cancer (CRC) (N=3), small cell lung cancer(SCLC) (N=2), and gastric cancer (GC) (N=2), with single cases ofesophageal adenocarcinoma (EAC), hormone-refractory prostate cancer(HRPC), non-small cell lung cancer (NSCLC), epithelial ovarian cancer(EOC), renal, tonsil, and urinary bladder cancers (UBC).

Immunohistology was performed on archival tissues from 17 patients, with13 (76.4%) having 2+ to 3+ membrane and cytoplasmic staining on >10% ofthe tumor cells in the specimens; 3 specimens (17.6%) were negative.Several representative cases are disclosed below.

All patients entered the trial with metastatic disease in sites typicalfor their primary cancer. CT determined that the median sum of thelargest tumor diameters for all patients was 9.7 cm (range 2.9 to 29.8cm), with 14 patients having 3 or more target lesions (over all patientsmedian=4, range 1-10 lesions) and a median of 2 non-target lesions(range=0-7 lesions) identified in their baseline studies. The mediannumber of prior systemic therapies was 3, with 7 patients (2 PDC and GC,1 each CRC, TNBC, tonsil) having one prior therapy, and 7 having five ormore prior therapies; eleven patients had prior radiation therapy. Priortopoisomerase I therapy was given to nine patients, with 2/3 CRC, 4/7PDC, and 1 patient with EAC receiving irinotecan, and 2/2 patients withSCLC having topotecan, with three of these (2 with SCLC and one withCRC) failing to respond to the anti-topoisomerase 1 therapy. Further,seven of 23 patients (2 undetermined) had responded to their last priortherapy, with a median duration of 3 months (range, 1-11 months).

Nearly all patients received multiple sacituzumab govitecan treatments(median, 10 doses) until there was definitive evidence of diseaseprogression by CT using RECIST 1.1; one patient withdrew becausesystematic deterioration, and 1 patient did not have their target lesionmeasured in first follow-up when a new lesion was observed.

TABLE 10 Baseline demographics and disease characteristics (N = 25patients). 8 mg/kg 10 mg/kg 12 mg/kg 18 mg/kg M/F 2/5 3/3 3/6 2/1 Age, yMedian (range) 52 (43-62) 58.5 (49-80)   60 (50-74) 56 (52-60) ECOGperformance status 0  3 1 2 0 1  1 5 7 3 Tumor Type N N N N Colorectal 21 0 0 Pancreas 3 1 3 0 TNBC 0 1 2 1 SCLC 0 0 1 1 Other^(a) 2 3 3 1 (EOC,GC) (GC, RCC, (UBC, (EAC) Tonsil) NSCLC, HRPC) Trop-2 expression N 1+ 1(TNBC) 2+ 3 (CRC) 3+ 10 (2 each of EAC, PDC, TNBC; 1 each of EOC,Tonsil, Negative NSCLC, SCLC) Not determined 3 (1 each of TNBC, Gastric,Renal) 8 (5 PDC, 1 each of HRPC, SCLC, UBC) Prior Therapy N N N NRadiotherapy 2 4 3 2 Systemic therapy^(c) 1  4 2 1 0 2  0 1 3 1 3  1 1 01 4  1 0 2 0 ≥5   1 2 3 1 Prior Topoisomerase I inhibitor 3 1 4 1 Tumormetastases (#patients) N N N N Target and non-target sitesChest/head/neck 0 2 4 3 Liver^(b) 4 (3)   4 (2)   5 (3)   2 (1)  Lungs^(b) 4 (3)   4 (2)   4 (3)   1 (1)   Lymph nodes 3 2 5 2Abdomen/pelvis 4 3 4 2 Bone 1 2 2 1 ≥3 target lesions 4 5 5 0 Patientstreated 7 6 9 3 Delay/adjustment 1^(st) cycle 1 0 5 2 Dose-limitingtoxicity 1^(st) cycle 0 0 0 2 #treatments at this dose median 3 (1-31)10 (1-31) 3 (1-8) 1 (1-2)  (range) 6 (3-31) 10 (1-31) 12 (4-34) 4 (3-16)Total # treatments median (range) ^(a)Other cancers include ovarian(EOC), gastric (GC), urinary bladder (UBC), non-small cell lung cancer(NSCLC), hormone refractory prostate cancer (HRPC), esophagealadenocarcinoma (EAC), renal cell cancer (RCC), and a squamous cellcarcinoma of the tonsil. ^(b)Number of patients with liver or lunginvolvement; in parenthesis number of these patients with both liver andlung involvement. ^(b)Systemic therapy includes chemotherapy and otherforms of therapy, including biologicals and investigational agents.

Dose Assessment—There were no dose delays or reductions, nor DLT eventsin the 3 patients (1 CRC, 2 PDC) enrolled at the starting dose level of8.0 mg/kg. At the next dose level of 12 mg/kg, nine patients wereenrolled because of protocol-required delays in administering the seconddose were encountered. Five patients experienced a delay in the firstcycle (4 had a 1-week delay, with 2 given myeloid growth factor support,and 1 patient having a 2-week delay before being given a second dose).All but 1 of these patients received 12 mg/kg as their second dose. Fourof the nine patients at the 12 mg/kg dose level had their third dosethat started the second cycle decreased to 9 mg/kg, and the second cyclewas delayed 1 additional week in 3 patients. Despite theseprotocol-required delays/reductions, none of the 9 patients had adose-limiting event during the first cycle (e.g., 1 patient haddisease-related grade 3 hemoglobin after first dose, 2 patients withgrade 3 neutropenia after first dose were given myeloid growth factors,1 had grade 3 neutropenia after first dose that recovered withoutsupport, 2 had grade 3 neutropenia after second dose, 2 patients hadgrade 2 neutropenia after the first or second dose, and 1 patient had noadverse events), and therefore accrual to the 18 mg/kg dose level wasallowed. Here, all three patients had dose delays after their firsttreatment, with only 1 patient receiving the second treatment at 18mg/kg. Two patients had dose-limiting grade 4 neutropenia, 1 after firstdose, the other after the second 18 mg/kg dose, with this latter patientalso experiencing grade 2 diarrhea after this dose. Therefore, with 0/9patients having DLT in the first cycle at 12 mg/kg, this level wasdeclared the MTD.

Additional dose-finding studies continued to refine the dose level thatwould allow multiple cycles to be given with minimal delay betweentreatments/cycles. Therefore, 4 more patients were enrolled at the 8mg/kg dose level, and a new intermediate level of 10 mg/kg was opened.Of the initial three patients enrolled at 8 mg/kg, two CRC patientscontinued treatment at 8 mg/kg for a total of 31 and 11 treatments,while a PDC patient received three 8 mg/kg doses before dose reductionto 6 mg/kg because of a grade-2 neutropenia on the fourth dose, and thencompleted 3 more treatments at this level before withdrawing due todisease progression. The additional 4 patients received 3 to 9 doses of8 mg/kg before withdrawing with disease progression. Two of thesepatients received only 1 dose before a protocol-required reduction to 6mg/kg, because of a grade-2 rash and grade-2 neutropenia.

Five of the six patients enrolled at 10 mg/kg received 6 to 30 doseswithout reduction before withdrawing due to disease progression. One GCpatient (# 9) developed grade 3 febrile neutropenia as well as grade 4hemoglobin after receiving 1 dose. While the febrile neutropenia wasconsidered possibly-related to treatment, because it occurred shortlyafter the first dose, a perforation in the stomach lining was found tolikely contribute to the grade 4 hemoglobin, and was consideredunrelated. Ultimately, the patient had rapid deterioration and died 4weeks from the first dose.

Thus, while the overall results supported 12 mg/kg as the MTD, since 8to 10 mg/kg were better tolerated in the first cycle and permittedrepeated cycles with minimal toxicity, Phase II clinical studies are inprogress to evaluate these 2 dose levels.

Adverse Events—There were 297 infusion of sacituzumab govitecan givenover 2-3 h, with most investigators electing to pre-medicate prior toeach infusion. There were no infusion-related adverse events. While morethan half of the patients experienced fatigue, nausea, alopecia,diarrhea, and neutropenia that were considered at least likely relatedto sacituzumab govitecan treatment; these were mostly grade 1 and 2(FIG. 16). The most reported grade 3 or 4 toxicity was neutropenia(N=8), but six of these patients were treated initially at 12 and 18mg/kg. Febrile neutropenia occurred in 2 patients, one was the GCpatient # 9 already mentioned who received only one 10 mg/kg dose, and asecond PDC patient (# 19), who had received 4 doses of 12 mg/kg.Diarrhea was mild in most patients, with only three (12%) experiencinggrade 3. Two occurred at the 12 mg/kg dose level, 1 after receiving 4doses, and the other after the first dose, but this patient received 6more doses at 12 mg/kg with only grade 2 diarrhea reported.Subsequently, both patients were prescribed an over-the-counteranti-diarrheal and treatment continued. There were no other significanttoxicities associated with sacituzumab govitecan, but two patientsreported a grade 2 rash and 3 patients had a grade 1 pruritus.

Efficacy—FIG. 17 (A) provides a graphic representation of the bestresponse as measured by the change in target lesions andtime-to-progression data from patients who had at least onepost-treatment CT measurement of their target lesions. Four patientswith disease progression are not represented in the graph, because theydid not have a follow-up CT assessment (N=1) or they had new lesions andtherefore progressed irrespective of their target lesion status (N=3).Overall, 3 patients had more than a 30% reduction in their targetlesions (partial response, PR). Two of these patients (# 3 and # 15) hadconfirmatory follow-up CTs, while the third patient (# 22) progressed atthe next CT performed 12 weeks later. Fifteen patients had stabledisease (SD), and 7 progressed (PD) as the best response by RECIST 1.1.The median time to progression from the start of treatment for 24patients (excluding 1 patient who received only 1 treatment andwithdrew) was 3.6 months [range, 1-12.8 months]; 4.1 months (range,2.6-12.8 months) for all patients with SD or PR (N=18). Of the ninepatients who received prior therapy containing a topoisomerase-Iinhibitor, two had significant reductions of their target lesions (28%and 38%), 5 had stable disease, including 2 for sustained periods (4.1and 6.9 months, respectively), whereas 2 progressed at their firstassessment.

FIG. 17 (B) compares TTP with survival of these patients, indicatingthat 16 patients survived from onset of therapy for 15-20 months,including two with a PR (patients 15 (TNBC) and 3 (CRC), and the otherfour with SD (2 CRC, 1 HRPC, 1 TNBC). Examples of radiological responsesin 2 patients with >30% reduction in their target lesions (PR) are shownin FIG. 18.

In addition to the 3 patients with PR as best response, there wereseveral notable cases of extended stable disease. A 50-year-old patientwith TNBC (patient 18; immunohistology Trop-2 expression=3+) experienceda 13% reduction after just 3 doses, culminating after 16 doses in a 19%reduction in the 4 target lesions (SLD decreased from 7.5 to 6.1 cm),before progressing 45 weeks after starting treatment and receiving 26doses. A 63-year-old female with CRC (patient 10; immunohistology 2+)with 7 prior treatments, including 3 separate courses of anirinotecan-containing regimens, had an overall 23% reduction in 5 targetlesions after receiving 5 doses of 10 mg/kg sacituzumab govitecan,culminating in a maximum 28% reduction after 18 doses. Her plasma CEAdecreased to 1.6 ng/mL from a baseline level of 38.5 ng/mL. Afterreceiving 25 doses (27 weeks), she had PD with a 20% increase from theSLD nadir. Interesting, plasma CEA at the time treatment ended was only4.5 ng/mL. A 68-year-old patient with HRPC (patient 20; noimmunohistology) presented with 5 target lesions (13.3 cm) and 5non-target lesions (3 bone metastases). He received 34 treatments over aperiod of 12.7 months until progression, with PSA levels increasinggradually over this time. Another notable case was a 52-year-old malewith esophageal cancer (patient 25; immunohistology 3+) who had received6 prior therapies, including 6 months of FOLFIRI as his 3^(rd) course oftreatment. Treatment was initiated at 18 mg/kg of sacituzumab govitecan,which was reduced to 13.5 mg/kg because of neutropenia. He had SD over aperiod of 30 weeks, receiving 15 doses before progressing. A 60-year-oldfemale with PDC (# 11) with liver metastases was treated at 10 mg/kg.Her baseline CA19-9 serum titer decreased from 5880 to 2840 units/mLafter 8 doses and there was disease stabilization (12% shrinkage as bestresponse) for a period or 15 weeks (11 doses) before a new lesion wasdiscovered. Nevertheless, because CA19-9 remained reduced (2814units/mL), the patient received another 8 treatments (3 months) at 10mg/kg before coming off study with progression of her target lesions.

At this time, the potential utility of testing Trop-2 expression inarchived samples from this small sampling of 16 patients with diversecancers is insufficient to allow for a definitive assessment, primarilybecause most showed elevated expression.

PK and immunogenicity—Concentrations of sacituzumab govitecan and IgG inthe 30-min serum sample are provided in Table 11, which showed a generaltrend for the values to increase as the dose increased. In arepresentative case, a patient with TNBC (# 15) received multiple doses,starting at 12 mg/kg, with subsequent reductions over the course of hertreatment. Concentrations of the IgG and sacituzumab govitecan in the30-min serum over multiple doses by ELISA were similar over time (notshown), adjusting lower when the dose was reduced. While residual IgGcould be found in the serum drawn immediately before the next dose(trough samples), no sacituzumab govitecan could be detected (notshown).

Total SN-38 concentration in the 30-min serum sample of patient 15 was3,930 ng/mL after the first dose in cycle 1 (C1D1), but when sacituzumabgovitecan treatment was reduced to 9.0 mg/kg for the second dose of thefirst cycle (C1D2), the level decreased to 2,947 ng/mL (not shown). Afurther reduction to 2,381 ng/mL was observed in the 6^(th) cycle, whenthe dose was further reduced to 6.0 mg/kg. The amount of free SN-38 inthese samples ranged from 88 to 102 ng/mL (2.4% to 3.6% of total SN-38),illustrating that >96% of the SN-38 in the serum in these peak sampleswas bound to IgG. Twenty-eight 30-min serum samples from 7 patients wereanalyzed by HPLC, with free SN-38 averaging 2.91±0.91% of the totalSN-38 in these samples. Free SN-38G concentrations measured in 4patients never exceeded SN-38 levels, and were usually several-foldlower. For example, patient # 25 had determinations assessed in the30-min sample for 12 injections over 8 cycles of treatment. At astarting dose of 18 mg/kg, he had 5,089 ng/mL of SN-38 in theacid-hydrolyzed sample (total SN-38) and just 155.2 ng/mL in thenon-hydrolyzed sample (free SN-38; 3.0%). Free SN-38G (glucuronidatedform) in this sample was 26.2 ng/mL, or just 14.4% of the total unboundSN-38+SN-38G in the sample. The patient continued treatment at 13.5mg/kg, with SN-38 averaging 3309.8±601.8 ng/mL in the 11 remaining peak,acid-hydrolyzed samples, while free SN-38 averaged 105.4±47.7 ng/mL(i.e., 96.8% bound to the IgG), and free SN-38G averaging 13.9±4.1 ng/mL(11.6% of the total SN-38+SN-38G). Importantly, in nearly all of thepatients, concentrations of SN-38G in the acid-hydrolyzed andnon-hydrolyzed samples were similar, indicating that none of the SN-38bound to the conjugate was glucuronidated.

TABLE 11 Serum concentration (μg/mL) of intact sacituzumab govitecan(ADC) and hRS7 IgG by ELISA. Assays were performed in samples taken 0.5h after the first dose. 8 mg/kg 10 mg/kg 12 mg/kg 18 mg/kg N 7 5 9 3 IgGADC IgG ADC IgG ADC IgG ADC Mean 193.1 141.5 203.35 185.77 239.2 183.3409.16 258.27 SD 56.5 23.8 55.72 54.14 70.7 71.8 88.78 143.26 Min 96.095.0 152.00 144.00 168.9 98.0 321.48 162.80 Max 249.0 169.3 285.85237.39 400.0 311.0 499.00 423.00

None of these patients had a positive baseline level (i.e., >50 ng/mL)or a positive antibody response to either the IgG or SN-38 over theircourse of treatment.

Discussion

Trop-2 is expressed abundantly in many epithelial tumors, making it anantigen of interest for targeted therapies (Cubas et al., 2009, BiochimBiophys Acta 1796:309-14), especially since it is considered aprognostic marker and oncogene in several cancer types (Cardillo et al.,2011, Clin Cancer Res 17:3157-69; Ambrogi et al., 2014, PLoS One9:e96993; Cubas et al., 2009, Biochim Biophys Acta 1796:309-14;Trerotola et al., 2013, Oncogene 32:222-33). Although its expression innormal tissues and relationship to another well-studied tumor-associatedantigen, EpCam, drew some initial words of caution regarding the safetyof developing immunotherapeutics to Trop-2 (Trerotola et al., 2009,Biochim Biophys Acta 1805:119-20), our studies in Cynomolgus monkeysthat express Trop-2 in tissues similar to humans indicated sacituzumabgovitecan was very well tolerated at a human equivalent dose of −40mg/kg (Cardillo et al., 2011, Clin Cancer Res 17:3157-69). At higherdoses, animals experienced neutropenia and diarrhea, known side-effectsassociated with SN-38 derived from irinotecan therapy, yet evidence forsignificant histopathological changes in Trop-2-expressing normaltissues was lacking (Cardillo et al., 2011, Clin Cancer Res 17:3157-69).Thus, with other preclinical studies finding sacituzumab govitecan waspotent at the low nanomolar level and effective in a variety of humanepithelial tumor xenografts at non-toxic doses, a phase I trial wasundertaken in patients who had failed one or more standard therapies fortheir diverse metastatic epithelial tumors.

A major finding of this study was that despite using a more conventionaldrug that is not considered as ultratoxic (drugs active in picomolarrange, whereas SN-38 has potency in the low nanomolar range), thesacituzumab govitecan anti-Trop-2-SN-38 conjugate proved clinically tobe therapeutically active in a wide range of solid cancers at doses withmoderate and manageable toxicity, thus exhibiting a high therapeuticindex. A total of 297 doses of sacituzumab govitecan were given to 25patients without incident; 4 patients received >25 injections.Importantly, no antibody response to the hRS7 IgG or SN-38 was detected,even in patients with multiple cycles of treatment for up to 12 months.Although Trop-2 is expressed in low quantities in a variety of normaltissues (Cardillo et al., 2011, Clin Cancer Res 17:3157-69), neutropeniawas the only dose-limiting toxicity, with myeloid growth factor supportused in 2 patients given ≥12 mg/kg of sacituzumab govitecan to expediterecovery and allow continuation of treatment in patients who hadexhausted their options for other therapy. While the MTD was declared tobe 12 mg/kg, 8.0 and 10.0 mg/kg dose levels were selected for furtherexpansion, since patients were more likely to tolerate additional cyclesat these levels with minimal supportive care, and responses wereobserved at these levels. Only 2 of 13 patients (15.4%) experiencedgrade-3 neutropenia at these dose levels. The grade 3 and 4 neutropeniaincidence for irinotecan monotherapy given weekly or once every 3 weeksin a front- or second-line setting was 14 to 26% (Camptosar—irinotecanhydrochloride injection, solution (prescribing information, packageinsert) Pfizer, 2012). With sacituzumab govitecan, only 1 patient at the10 mg/kg dose level had grade-3 diarrhea. This incidence is lower thanthe 31% of patients given weekly×4 doses of irinotecan who experiencedgrades 3 and 4 late diarrhea (Camptosar—irinotecan hydrochlorideinjection, solution (prescribing information, package insert) Pfizer,2012). Other common toxicities attributed to sacituzumab govitecanincluded fatigue, nausea, and vomiting, most being grade 1 and 2, aswell as alopecia. Two incidents of febrile neutropenia and one of grade3 deep vein thrombosis also occurred at the 10 and 12 mg/kg dose levels.UGT1A1 monitoring was not initiated until after dose exploration wascompleted, and therefore an assessment of its contribution to toxicitycannot be reported at this time.

Patients enrolled in this trial were not pre-selected for Trop-2expression, primarily because immunohistological assessments of tissuemicroarrays of diverse cancers (such as prostate, breast, pancreas,colorectal, and lung cancers) had indicated the antigen was presentin >90% of the specimens (not shown). In addition, Trop-2 was not foundin the sera of 12 patients with diverse metastatic cancers, furthersuggesting that a serum assay would not be useful for patient selection.Although we are attempting to collect archival specimens of the tumorsfrom patients enrolled in the trial, there is insufficient evidence atthis time to suggest patient selection based on immunohistologicalstaining will correlate with anti-tumor activity, so no patientenrichment based on Trop-2 expression has been undertaken.

As a monotherapy, sacituzumab govitecan had good anti-tumor activity inpatients with diverse metastatic, relapsed/refractory, epithelialtumors, showing appreciable reductions in target lesions by CT, usingRECIST1.1 criteria, including sustained disease stabilization. Three(12%) of the 25 patients (1 each of SCLC [after progressing withtopotecan], TNBC, and colon cancer) had >30% reductions of their targetlesions before progressing 2.9, 4.3, and 7.1 months, respectively, fromthe onset of therapy. Fifteen patients (60%) had SD, with 9 of theseprogressing after >4 months from the start of treatment. Responses ordisease stabilization occurred in 7 of 9 patients who had prior therapywith a topoisomerase I inhibitor-containing drug or regimen. Three ofthese failed to respond to their prior topoisomerase I inhibitor therapy(irinotecan or topotecan), yet sacituzumab govitecan was able to inducetumor shrinkage in 2 of them: 13% in a patient with colon cancer and 38%in the other with SCLC. Thus, sacituzumab govitecan may betherapeutically active in those who failed or relapsed to a priortopoisomerase I-containing regimen, which should be examined further inthe Phase II expansion study.

Although the largest number of patients enrolled in this trial hadadvanced pancreatic ductal cancer (N=7; median time to progression 2.9months]; range, 1.0 to 4.0 months), even in this difficult-to-treatdisease, there have been encouraging reductions in target lesions andCA19-9 serum concentrations to suggest activity (Picozzi et al., 2014,presented at the AACR Special Conference “Pancreatic Cancer: Innovationsin Research and Treatment, New Orleans, La. USA, p. B99). However,responses in patients with TNBC and SCLC are of particular interest,given the need for targeted therapies in these indications. Indeed,additional partial responses in patients with TNBC (Goldenberg et al.,2014, presented at the AACR San Antonio Breast cancer Symposium, SanAntonio, Tex.) and SCLC (Goldenberg et al., 2014, Sci Transl Med)observed in the on-going expansion phase of this trial have suggestedfurther emphasis on these cancers, but encouraging responses in NSCLC,EAC, UBC, and CRC are also being followed. Indeed, in a recent update ofthe on-going trial of sacituzumab govitecan, in 17 TNBC patients studiedto date, an overall response rate (PR) of 29%, with 46% clinical benefitrate (PR+SD >6 months) has been observed. Long-term survival (15-20months) was observed for almost 25% (6/25) of the patients studied, andincluded 2 with PRs and 4 with SD, including patients with TNBC (N=2),CRC (N=3), and HRPC (N=1).

Analysis of the serum samples 30 min after the end of infusionshowed >96% of the SN-38 was bound to the IgG. More detailedpharmacokinetics will be available when the phase II portion of thetrial is completed. HPLC analysis also detected only trace amounts offree SN-38G in the serum, whereas with irinotecan therapy the AUC forthe less active SN-38G is >4.5-fold higher than SN-38 (Xie et al., 2002,J Clin Oncol 20:3293-301). Comparison of SN-38 delivery in tumor-bearinganimals given sacituzumab govitecan and irinotecan has indicated theSN-38 bound to the IgG is not glucuronidated, whereas in animals givenirinotecan, >50% of the total SN-38 in the serum is glucuronidated(Goldenberg et al., 2014, J Clin Oncol 32:Abstract 3107). Moreimportantly, analysis of SN-38 concentrations were ˜135-fold higher inCapan-1 human pancreatic cancer xenografts given sacituzumab govitecanthan irinotecan (Goldenberg et al., 2014, Sci Transl Med). Thus,sacituzumab govitecan has several distinct advantage over non-targetedforms of topoisomerase-I inhibitors: (i) a mechanism that selectivelyretains the conjugate in the tumor (anti-Trop-2 binding), and (ii) thetargeted SN-38 also appears to be fully protected (i.e., notglucuronidated and in the lactone form), such that any SN-38 accreted bythe tumor cells either by the direct internalization of the conjugate orthrough its release into the tumor microenvironment from the conjugatebound to the tumor will be in its most potent form. These resultssuggest that a moderately-toxic, but well understood, cytotoxic agent,SN-38, can be effective as part of a tumor-targeting ADC, such assacituzumab govitecan. But by administering an ADC with amoderately-toxic drug conjugated at a high drug:antibody ratio (7.6:1),higher concentrations of SN-38 can be delivered to the cancers targeted,as suggested in the improved concentration of SN-38 achieved withsacituzumab govitecan compared to that released from irinotecan.

In conclusion, this phase I experience has shown that sacituzumabgovitecan was tolerated with moderate and manageable toxicity, allrelated to the activity of SN-38, with no evidence of damage to normaltissues known to contain Trop-2. Importantly, sacituzumab govitecan wasactive in patients with diverse metastatic solid tumors, even afterfailing prior therapy with topoisomerase-I inhibitors. Thus, it appearsfrom this initial experience that sacituzumab govitecan has a hightherapeutic index, even in patients with tumors not known to beresponsive to topoisomerase I inhibitors, such as SCLC and TNBC. Thisclinical trial is continuing, focusing on starting doses of 8 and 10mg/kg in patients with TNBC, SCLC, and other Trop-2⁺ cancers.

Example 15. Use of IMMU-132 in Triple Negative Breast Cancer (TNBC)

The Trop-2/TACSTD2 gene has been cloned (Fornaro et al., 1995, Int JCancer 62:610-18) and found to encode a transmembrane Ca⁺⁺-signaltransducer (Basu et al., 1995, Int J Cancer 62:472-72; Ripani et al.,1998, Int J Cancer 76:671-76) functionally linked to cell migration andanchorage-independent growth, with higher expression in a variety ofhuman epithelial cancers, including breast, lung, gastric, colorectal,pancreatic, prostatic, cervical, head-and-neck, and ovarian carcinomas,compared to normal tissues (Cardillo et al., 2011, Clin Cancer Res17:3157-69; Stein et al., 1994; Int J Cancer Suppl 8:98-102; Cubas etal., 2009, Biochim Biophys Acta 196:309-14; Trerotola et al., 2013,Oncogene 32:222-33). The increased expression of Trop-2 has beenreported to be necessary and sufficient for stimulation of cancer growth(Trerotola et al., 2013, Oncogene 32:222-33), while a bi-cistroniccyclin D1-Trop-2 mRNA chimera is an oncogene (Guerra et al., 2008,Cancer Res 68:8113-21). Importantly, elevated expression has beenassociated with more aggressive disease and a poor prognosis in severalcancer types (Cubas et al., 2009, Biochim Biophys Acta 196:309-14;Guerra et al., 2008, Cancer Res 68:8113-21; Bignotti et al., 2010, Eur JCancer 46:944-53; Fang et al., 2009, Int J Colorectal Dis 24:875-84;Muhlmann et al., 2009, J Clin Pathol 62:152-58), including breast cancer(Ambrogi et al., 2014, PLoS One 9:e96993; Lin et al., 2013, Exp MolPathol 94:73-8). Increased Trop-2 mRNA is a strong predictor of poorsurvival and lymph node metastasis in patients with invasive ductalbreast cancers, and Kaplan-Meier survival curves showed that breastcancer patients with high Trop-2 expression had a significantly shortersurvival (Lin et al., 2013, Exp Mol Pathol 94:73-8).

Methods

DAR determination by HIC—Clinical lots of IMMU-132 were analyzed byhydrophobic interaction chromatography (HIC) using a butyl-NPR HPLCcolumn (Tosoh Bioscience, King of Prussia, Pa.). IMMU-132 injections(100 μg) were resolved with a 15-min linear gradient of 2.25-1.5 M NaClin 25 mM sodium phosphate, pH 7.4, run at 1 mL/min and room temperature.

DAR determination by LC-MS—Because the interchain disulfides are reducedand the resulting sulfhydryl groups are used for drug conjugation (orblocked), the heavy and light chains resolved during LC-MS analysiswithout addition of reducing agents, and were analyzed independently.Different lots of IMMU-132 were injected on an Agilent 1200 series HPLCusing an Aeris Widepore C4 reverse-phase HPLC column (3.6 50×2.1 mm) andresolved by reverse phase HPLC with a14-min linear gradient of 30-80%acetonitrile in 0.1% formic acid. Electrospray ionization time of flight(ESI-TOF) mass spectrometry was accomplished with an in-line Agilent6210 ESI-TOF mass spectrometer with Vcap, fragmentor and skimmer set to5000V, 300V and 80V, respectively. The entire RP-HPLC peak representingall kappa or heavy chain species were used to generate deconvoluted massspectra.

Cell lines—All human cancer cell lines used in this study were purchasedfrom the American Type Culture Collection (Manassas, Va.), except wherenoted, and all were authenticated by short tandem repeat (STR) assay bythe ATCC.

Trop-2 surface expression on various human breast carcinoma celllines—Expression of Trop-2 on the cell surface is based on flowcytometry. Briefly, cells were harvested with Accutase Cell DetachmentSolution (Becton Dickinson (BD); Franklin Lakes, N.J.; Cat. No. 561527)and assayed for Trop-2 expression using QuantiBRITE PE beads (BD Cat.No. 340495) and a PE-conjugated anti-Trop-2 antibody (eBiosciences, Cat.No. 12-6024) following the manufacturer's instructions. Data wereacquired on a FACSCalibur Flow Cytometer (BD) with CellQuest Prosoftware, with analysis using Flowjo software (Tree Star; AshlandOreg.).

In vitro cytotoxicity testing—Sensitivity to SN-38 was determined usingthe3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazoliumdye reduction assay (MTS dye reduction assay; Promega, Madison, Wis.).Briefly, cells were plated into 96-well clear, flat-bottomed plates asdescribed above. SN-38 dissolved in DMSO was diluted with media to afinal concentration of 0.004 to 250 nM. Plates were incubated inhumidified chamber for 96 h 37° C./5% CO₂, after which the MTS dye wasadded and placed back into the incubator until untreated control cellshad an absorbance greater than 1.0. Growth inhibition was measured as apercent of growth relative to untreated cells. Dose-response curves weregenerated from the mean of triplicate determinations, and IC₅₀-valueswere calculated using Prism GraphPad Software.

In vitro specificity testing by flow cytometry with rH2AX-stainedcells—For drug activity testing, HCC1806 and HCC1395 TNBC cell linescells were seeded in 6-well plates at 5×10⁵ cells/well and held at 37°C. overnight. After cooling the cells for 10 min on ice, the cells wereincubated with either IMMU-132 or hA20 anti-CD20-SN38 at ˜20 μg/ml(equal SN38/well for both agents) for 30 minutes on ice, washed threetimes with fresh media, and then returned to 37° C. overnight. Cellswere trypsinized briefly, pelleted by centrifugation, fixed in 4%formalin for 15 min, then washed and permeabilized in 0.15% Triton-X100in PBS for another 15 min. After washing twice with 1% bovine serumalbumin-PBS, cells were incubated with mouse anti-rH2AX-AF488 (EMDMillipore Corporation, Temecula, Calif.) for 45 minutes at 4° C. Thesignal intensity of rH2AX was measured by flow cytometry using a BDFACSCalibur (BD Biosciences, San Jose, Calif.).

IHC of Trop-2 in tumor microarrays and patient specimens—This involvedstandard IHC methods on tissue and microarray sections. Scoring wasbased on the intensity of the stain in >10% of the tumor cells withinthe specimen, including negative, 1+ (weak), 2+ (moderate), and 3+(strong).

In vivo therapeutic studies in xenograft models—SN-38 equivalents in adose of 250 μg ADC to a 20-gram mouse (12.5 mg/kg) is equal to 0.2 mgSN-38/kg. For irinotecan (irinotecan-HCl injection; AREVAPharmaceuticals, Inc., Elizabethtown, Ky.), 10 mg irinotecan/kg convertsto 5.8 mg SN-38/kg based on mass.

Immunoblotting—Cells (2×10⁶) were plated in 6-well plates overnight. Thefollowing day they were treated with either SN-38 or IMMU-132 at anSN-38 concentration equivalent of 0.4 μg/mL (1 μM) for 24 and 48 h.Parental hRS7 was used as a control for the ADC.

Quantification of SN-38 in mice with human tumor xenografts—Two groups,each with 15 animals bearing subcutaneous implants of the humanpancreatic carcinoma cell line, were administered either irinotecan orIMMU-132. At 5 different intervals, 3 animals per group were euthanized.The Capan-1 tumors (0.131±0.054 g; N=30) were removed and homogenized indeionized water (DI) (1 part tissue+10 parts DI water); serum wasdiluted with an equal part DI water. Serum and tissue homogenates wereextracted and analyzed by reversed-phase HPLC (RP-HPLC). While extractedsamples were adequate for detecting products from the irinotecan-treatedanimals, samples from animals given IMMU-132 were split into 2 portions,with one undergoing an acid-hydrolysis step in order to release all ofthe SN-38 bound to the IgG, which would otherwise go undetected in theextracted samples.

Statistics—Statistical analyses were performed using GraphPad Prismversion 5.00 for Windows, GraphPad Software, La Jolla Calif. USA. Thespecific testing performed is identified with each study.

Results

SN-18 structure and properties—IMMU-132 utilizes the topoisomerase Iinhibitor, SN-38, the water soluble metabolite of the anticancercamptothecin, irinotecan(7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin),that is therapeutically active in colorectal, lung, cervical, andovarian cancers (Garcia-Carbonero et al., 2002, Clin Cancer Res8:641061). An important advantage for selecting SN-38 is that the drug'sin-vivo pharmacology is well known. Irinotecan must be cleaved byesterases to form SN-38, which is 2-3 orders of magnitude more potentthan irinotecan, with activity in the low nanomolar range (Kawato etal., 1991, Cancer Res 51:4187-91). At physiological pH, camptothecinsexist in an equilibrium comprising the more active lactone form and theless active (10% potency) open carboxylic acid form (Burke & Mi, 1994, JMed Chem 37:40-46).

The design of the SN-38 derivative used in IMMU-132, CL2A-SN-38,addressed multiple challenges in using this drug in the ADC format, andinvolved the following features: (i) A short polyethylene glycol (PEG)moiety was placed in the cross-linker to confer aqueous solubility tothis highly insoluble drug; (ii) a maleimide group was incorporated forfast thiol-maleimide conjugation to mildly reduced antibody, with aspecially-designed synthetic procedure enabling high-yield incorporationof maleimide in the context of assembling the carbonate linkage; (iii) abenzylcarbonate site provided a pH-mediated cleavage site to release thedrug from the linker; and (iv) importantly, the crosslinker was attachedto SN-38's 20-hydroxy position, which kept the lactone ring of the drugfrom opening to the less active carboxylic acid form under physiologicalconditions (Giovanella et al., 2000, Ann NY Acad Sci 922:27-35). Thesynthesis of SN-38 derivatives and the conjugation of CL2A-SN-38 tomildly reduced hRS7 IgG has been described above. The limited reductionprocedure breaks only the interchain disulfide bridges between theheavy-heavy and heavy-light chains, but not the intra-domain disulfides,generating 8 site-specific thiols per antibody molecule. It is thenconjugated to CL2A-SN-38, purified by diafiltration, and lyophilized forstorage. During manufacturing, conditions are adjusted to minimize anyloss of SN-38 from IMMU-132, with the final lyophilized productconsistently having <1% free SN-38 when reconstituted. However, whenplaced in serum and held at 37° C., SN-38 is released from the conjugatewith a half-life of ˜1 day (not shown).

The release of SN-38 appears to be an important feature of IMMU-132,with this type of linker selected based on efficacy studies that testedSN-38 conjugated to a variety of linkers that had different rates ofSN-38 release, from ˜10 h release half-life to being highly stable (Moonet al., 2008(30, 31). Optimal therapeutic activity was found with aconjugate having an intermediate release rate in serum of ˜2 days. Wesubsequently improved the manufacturing process for this type of linker,designated CL2A, by removing a phenylalanine residue (Cardillo et al.,2011, Clin Cancer Res 17:3157-64, and then again compared the efficacywith that of another stably-linked anti-Trop-2 conjugate (CL2) that wasdesigned to release SN-38 only under lysosomal conditions (i.e., in thepresence of cathepsin B and pH 5.0). In animal models, the anti-Trop-2conjugate prepared with the CL2A linker yielded better therapeuticresponses than when SN-38 was linked stably, indicating that evenantibodies that internalized quickly benefitted when SN-38 was allowedto be released in serum with a half-life of ˜1 day (Govidan et al. 2013,Mol Cancer Ther 12:968-78). Since clinical studies with radiolabeledantibodies have found the antibodies localize in tumors within a fewhours, reaching peak concentrations within 1 day (Sharkey et al., 1995,Cancer Res 55:5935s-45s), selectively enhanced concentrations of SN-38are delivered locally in the tumor through internalization of the intactconjugate, extracellular release of the free drug, or both mechanisms inconcert.

Drug-antibody ratio (DAR) determination. Five clinical lots of IMMU-132were evaluated by hydrophobic interaction HPLC (HIC-HPLC), whichresolved three peaks representing species with DARs of 6, 7 and 8, withthe greatest fraction comprising a DAR=8 (not shown). IMMU-132 wasproduced consistently by this manufacturing process, with an overall DAR(DARAvE) of 7.60±0.03 among the five clinical lots (not shown). HIC-HPLCresults were confirmed by liquid chromatography-mass spectrometry(LC-MS) (not shown). The analysis showed that >99% of the 8 availablesulfhydryl groups were coupled with the CL2A linker, either with orwithout SN-38. There were no unsubstituted (or N-ethylmaleimide capped)heavy or light chains detected. Thus, the difference in DAR among thespecies results from SN-38 liberation from the linker duringmanufacturing and not from a lower initial substitution ratio. Onceprepared and lyophilized, IMMU-132 has been stable for several years.

Effect of DAR on pharmacokinetics and anti-tumor efficacy in mice. Micebearing Trop-2⁺ human gastric carcinoma xenografts (NCI-N87) were given2 treatments 7 days apart, each with equal protein (0.5 mg) doses ofIMMU-132 having DARs of 6.89, 3.28, or 1.64 (FIG. 19 (A)). Animalstreated with the ADCs having a DAR of 6.89 had a significantly improvedmedian survival time (MST) compared to mice given ADCs with either 3.38or 1.64 DARs (MST=39 days vs. 25 and 21 days, respectively; P<0.0014).There was no difference between groups treated with the 3.28 or 1.64 DARconjugates and the saline control group.

To further elucidate the importance of a higher DAR, mice bearingNCI-N87 gastric tumors were administered 0.5 mg IMMU-132 with a DAR of6.89 twice weekly for two weeks (FIG. 19 (B)). Another group receivedtwice the protein (1 mg) dose of an IMMU-132 conjugate with a DAR of3.28. Although both groups received the same total amount of SN-38 (36μg) with each dosing scheme, those treated with the 6.89 DAR conjugateinhibited tumor growth significantly more than tumor-bearing animalstreated with the 3.28 DAR conjugate (P=0.0227; AUC). Additionally,treatment with the lower DAR was not significantly different than theuntreated controls. Collectively, these studies indicate that a lowerDAR reduces efficacy.

An examination of the pharmacokinetic behavior of conjugates prepared atthese different ratios was performed in non-tumor-bearing mice given 0.2mg of each conjugate, unconjugated hRS7 IgG, or hRS7 IgG that wasreduced and then capped with N-ethylmaleimide. Serum was taken at 5intervals from 0.5 to 168 h and assayed by ELISA for hRS7 IgG. There wasno significant difference in the clearance of these conjugates comparedto the unconjugated IgG (not shown). Thus, the substitution level didnot affect the pharmacokinetics of the conjugates, and equallyimportant, the reduction of the interchain disulfide bonds did notappear to destabilize the antibody.

Trop-2 expression in TNBC and SN-38 sensitivity. Trop-2 expression wasdetermined by immunohistochemistry (IHC) in several tissue microarraysof human tumor specimens. In one microarray containing 31 TNBCspecimens, as well as 15 hormone-receptor- or HER-2-positive breastcancers, positive staining occurred in over 95% of the tumors, with3+staining indicated in 65% of the cases.

Table 12 lists 6 human breast cancer cell lines, including four TNBC,showing their surface expression of Trop-2 and sensitivity to SN-38.Trop-2 surface expression in 5 of the 6 cell lines exceeded 90,000copies per cell. SN-38 potency ranged from 2 to 6 nM in 5 of the 6 celllines, with MCF-7 having the lowest sensitivity of 33 nM. In vitropotency for IMMU-132 is not provided, because nearly all of the SN-38associated with IMMU-132 is released into the media during the 4-dayincubation period, and therefore its potency would be similar to that ofSN-38. Therefore, a different strategy was required to illustrate theimportance of antibody targeting as a mechanism for delivering SN-38.

TABLE 12 Trop-2 expression and SN-38 sensitivity in breast cancer celllines. Receptor Trop-2 surface IC₅₀ (nM) Cell Line status expression^(A)SN-38 SK-BR-3 BERT⁺ 328,281 ± 47,996 2 MDA-MB-468 TNBC 301,603 ± 29,4702 HCC38 TNBC 181,488 ± 69,351 2 MCF-7 ER⁺ 110,646 ± 17,233 33 HCC1806TNBC  91,403 ± 20,817 1 MDA-MIB-231 TNBC 32,380 ± 5,460 6 ^(A)Mean ± SDnumber of surface Trop-2 molecules per cell from three separate assays.

Antigen-positive (HCC1806) or -negative (HCC1395) TNBC cell lines thatwere incubated at 4° C. for 30 min with either IMMU-132 or a non-bindinganti-CD20 SN-38 conjugate. The cells were then washed to remove unboundconjugate, and then incubated overnight at 37° C. Cells were fixed andpermeabilized, and then stained with the fluorescentanti-phospho-histone H2A.X antibody to detect dsDNA breaks by flowcytometry (Bonner et al., 2008, Nat Rev Cancer 8:957-67) (Table 13). TheTrop-2⁺ breast cancer cell line, HCC1806, when incubated with IMMU-132,had an increase in median fluorescence intensity (MFI) from 168(untreated baseline) to 546, indicating the increased presence of dsDNAbreaks, whereas the MFI for cells incubated with the non-bindingconjugate remained at baseline levels. In contrast, MFI for the Trop-2antigen-negative cell line, HCC1395, remained at baseline levelsfollowing treatment with either IMMU-132 or the non-binding controlconjugate. Thus, the specificity of IMMU-132 over an irrelevant ADC wasconclusively revealed by evidence of dsDNA breaks only inTrop-2-expressing cells incubated with the anti-Trop-2-bindingconjugate.

TABLE 13 Specificity of IMMU-132 anti-tumor activity in vitro using flowcytometry with phospho-H2AX (anti-histone)-stained cells.^(A) Medianfluorescence intensity HCC1806 HCC1395 Treatment (Trop-2⁺) (Trop-2⁻)Cell alone 4.25 5.54 Cell + anti-rH2AX-AF488 168 122 Cell + IMMU-132 +anti-rH2AX-AF488 546 123 Cell + hA20-SN38 + anti-rH2AX-AF488 167 123^(A)HCC1806 (Trop-2⁺) or HCC1395 (Trop-2⁻) were incubated at 4° C. withIMMU-132 or a non-binding control conjugate (anti-CD20-SN-38) for 30min, washed and incubated overnight at 37° C. in fresh drug-free media.Cells were harvested, fixed, and permeabilized, then stained with thefluorescently-conjugated anti-histone antibody (rH2AX-AF488) fordetection of double-stranded DNA breaks. The median fluorescenceintensity (MFI) is given for (a) background staining of the cells alone(no anti-histone antibody), (b) the background level of dsDNA breaks forthe cells that had no prior exposure to the conjugates, and (c) afterexposed to IMMU-132 or hA20-SN-38 conjugates.

In vivo efficacy of hRS7-CL2A-SN-38 in TNBC xenografts. The efficacy ofIMMU-132 was assessed in nude mice bearing MDA-MB-468 TNBC tumors (FIG.20 (A)). IMMU-132 at a dose of 0.12 or 0.20 mg/kg SN-38-equivalents(0.15 and 0.25 mg IMMU-132/dose) induced significant tumor regression,compared to saline, irinotecan (10 mg/kg; ˜5.8 mg/kg SN-38 equivalentsby weight), or a control anti-CD20 ADC, hA20-CL2A-SN-38, given at thesame 2 dose levels (P<0.0017, area under the curve, AUC). Since miceconvert irinotecan to SN-38 more efficiently than humans (38) (in ourstudies, it averaged ˜25%, see below), at this irinotecan dose ˜145 to174 μg of SN-38 would be produced, while the administered dose ofIMMU-132 contained only 9.6 μg. Nevertheless, because IMMU-132selectively targeted SN-38 to the tumors, it was more efficacious. Theseresults corroborate findings in other solid tumor models (Cardillo etal., 2011, Clin Cancer Res 17:3157-69) showing that specific targetingof a small amount of SN-38 to the tumor with IMMU-132 is much moreeffective than a much larger dose of irinotecan, or for that matter amixture of hRS7 IgG with an equal amount of free SN-38 (Cardillo et al.,2011, Clin Cancer Res 17:3157-69). The unconjugated RS7 antibody, evenat repeated doses of 1 mg per animal, did not show any antitumor effects(Cardillo et al., 2011, Clin Cancer Res 17:3157-69). However, in-vitrostudies with gynecological cancers expressing Trop-2 have indicated cellkilling with the RS7 mAb by antibody-dependent cellular cytotoxicity(Bignotti et al., 2010, Eur J Cancer 46:944-53; Raji et al., 2011, J ExpClin Cancer Res 30:106; Varughese et al., 2011, Gynecol Oncol 122:171-7;Varughese et al., 2011, Am J Obstet Gyneol 205:567). Also, a monovalentFab of another anti-Trop-2 antibody has been reported to betherapeutically active in vitro and in animal studies.

On therapy day 56, four of the seven tumors in mice given 0.12 mg/kg ofthe hA20-CL2A-SN-38 control ADC already had progressed to the endpointof 1.0 cm³ (FIG. 20 (B)). At this time, these animals were treated withIMMU-132, electing to use the higher dose of 0.2 mg/kg in an attempt toaffect the progression of these much larger tumors. Despite thesubstantial size of the tumors in several animals, all mice demonstrateda therapeutic response, with tumors significantly smaller in size fiveweeks later (total volume [TV]=0.14±0.14 cm³ vs. 0.74±0.41 cm³,respectively; P=0.0031, two-tailed t-test). Similarly, we chose twoanimals in the irinotecan-treated group with tumors that progressed to˜0.7 cm³ and re-treated one with irinotecan and the other with IMMU-132(not shown). Within 2 weeks of ending treatment, the tumor in theirinotecan-treated animal decreased 23% and then began to progress,while the tumor treated with IMMU-132 had stabilized with a 60% decreasein tumor size. These results demonstrate that even in tumors thatcontinued to grow after exposure to SN-38 via a non-specific ADC, asignificantly enhanced therapeutic response could be achieved whentreated with the Trop-2-specific IMMU-132. However, specific therapeuticeffects with IMMU-132 were not achieved in MDA-MB-231 (FIG. 20 (C)).This cell line had the lowest Trop-2 levels, but also was the leastsensitive to SN-38.

Mechanism of action of IMMU-132 in TNBC—The apoptotic pathway utilizedby IMMU-132 was examined in the TNBC cell line, MDA-MB-468, and in theHER2⁺ SK-BR-3 cell line, in order to confirm that the ADC functions onthe basis of its incorporated SN-38 (not shown). SN-38 alone andIMMU-132 mediated >2-fold up-regulation of p21^(WAF1/Cip1) within 24 hin MDA-MB-468, and by 48 h, the amount of p21^(WAF1/Cip1) in these cellsbegan to decrease (31% and 43% with SN-38 or IMMU-132, respectively).Interestingly, in the HER2⁺ SK-BR-3 tumor line, neither SN-38 norIMMU-132 mediated the up-regulation of p21^(WAF1/Cip1) aboveconstitutive levels in the first 24 h, but as seen in MDA-MB-468 cellsafter 48-h exposure to SN-38 or IMMU-132, the amount of p21^(WAF1/Cip1)decreased >57%. Both SN-38 and IMMU-132 resulted in cleavage ofpro-caspase-3 into its active fragments within 24 h, but with thegreater degree of active fragments observed after exposure for 48 h. Ofnote, in both cell lines, IMMU-132 mediated a greater degree ofpro-caspase-3 cleavage, with the highest level observed after 48 h whencompared to cells exposed to SN-38. Finally, SN-38 and IMMU-132 bothmediated poly ADP ribose polymerase (PARP) cleavage, starting at 24 h,with near complete cleavage after 48 h. Taken together, these resultsconfirm that IMMU-132 has a mechanism of action similar to that of freeSN-38 when administered in vitro.

Delivery of SN-38 by IMMU-132 vs. irinotecan in a human tumor xenograftmodel -Constitutive products derived from irinotecan or IMMU-132 weredetermined in the serum and tumors of mice implanted s.c. with a humanpancreatic cancer xenograft (Capan-1) administered irinotecan (773 μg;SN-38 equivalents=448 μg) and IMMU-132 (1.0 mg; SN-38 equivalents=16μg).

Irinotecan cleared very rapidly from serum, with conversion to SN-38 andSN-38G seen within 5 min. None of the products was detected at 24 h. TheAUCs over a 6-h period were 21.0, 2.5, and 2.8 μg/mL·h for irinotecan,SN-38, and SN-38G, respectively (SN-38 conversion inmice=[2.5+2.8)/21=25.2%]). Animals given IMMU-132 had much lowerconcentrations of free SN-38 in the serum, but it was detected through48 h (FIG. 5A). Free SN-38G was detected only at 1 and 6 h, and was 3-to 7-times lower than free SN-38.

In the Capan-1 tumors excised from irinotecan-treated animals,irinotecan levels were high over 6 h, but undetectable a 24 h(AUC_(5 min-6 h)=48.4 μg/g·h). SN-38 was much lower and detected onlythrough 2 h (i.e., AUC_(5 min-2 h)=0.4 μg/g·h), with SN-38G valuesalmost 3-fold higher (AUC=1.1 μg/g·h) (not shown). Tumors taken fromanimals given IMMU-132 did not have any detectable free SN-38 or SN-38G,but instead, all SN-38 in the tumor was bound to IMMU-132. Importantly,since no SN-38G was detected in the tumors, this suggests SN-38 bound toIMMU-132 was not glucuronidated. The AUC for SN-38 bound to IMMU-132 inthese tumors was 54.3 μg/g·h, which is 135-fold higher than the amountof SN-38 in the tumors of animals treated with irinotecan over the 2-hperiod that SN-38 could be detected, even though mice given irinotecanreceived 28-fold more SN-38 equivalents than administered with IMMU-132(i.e., 448 vs 16 μg SN-38 equivalents, respectively)

Discussion

We describe a new ADC targeting Trop-2, and early clinical resultssuggest it is well tolerated and effective in patients with TNBC, aswell as other Trop-2⁺ cancers (Bardia et al., 2014, San Antonio BreastCancer Symposium, P5-19-27). Due to its distinct properties, IMMU-132represents a second-generation ADC. Typically, ADCs require 4 broadattributes to be optimally-effective: (i) selective targeting/activity;(ii) binding, affinity, internalization, and immunogenicity of theantibody used in the ADC; (iii) the drug, its potency, metabolism andpharmacological disposition, and (iv) how the drug is bound to theantibody. Target selectivity is the most common requirement for allADCs, since this will play a major role in defining the therapeuticindex (ratio of toxicity to tumor vs. normal cells). Trop-2 appears tohave both a high prevalence on a number of epithelial cancers, but it isalso expressed by several normal tissues (Cubas et al., 2009, BiochimBiophys Acta 1796:309-14; Trerotola et al., 2013, Oncogene 32:222-33;Stepan et al., 2011, 59:701-10), which could have impacted specificity.However, expression in normal tissues appears to be lower than incancers (Bignotti et al., 2010, Eur J Cancer 46:944-53), and Trop-2appears to be shielded by normal tissue architecture that limitsaccessibility to an antibody, whereas in cancer, these tissue barriersare compromised by the invading tumor. Evidence of this was apparentfrom initial toxicological studies in monkeys, where despite escalatingIMMU-132 doses to levels leading to irinotecan-like neutropenia anddiarrhea, histopathological damage to Trop-2-expressing normal tissuesdid not occur (Cardillo et al., 2011, Clin Cancer Res 17:3157-69). Theseresults appear to have been confirmed clinically, where no specificorgan toxicity has been noted in patients to-date, except for the knowntoxicities of the parental compound, irinotecan (Bardia et al., 2014,San Antonio Breast Cancer Symposium, P5-19-27), which are moremanageable with IMMU-132.

A generally accepted and important criterion for ADC therapy is that theantibody should internalize, delivering its chemotherapeutic inside thecell, where it is usually metabolized in lysosomes. Despite IMMU-132'sinternalization, we believe that the linker in this ADC, which affordslocal release of SN-38 that likely can induce a bystander effect on thecancer cells, is another feature that sets this platform apart fromthose using an ultratoxic drug. Indeed, having an ultratoxic agentlinked stably to the IgG is the only configuration that would preserve auseful therapeutic window for those types of compounds. However, using amore moderately-toxic drug does not give the latitude to use a linkerthat would release the drug too early once in the circulation. Our groupexplored linkers that released SN-38 from the conjugate with differenthalf-lives in serum, ranging from ˜10 h to a highly stable linker, butit was the linker with the intermediate stability that provided the besttherapeutic response in mouse-human tumor xenograft models (Moon et al.,2008, J Med Chem 51:6916-26; Govindan et al., 2009, Clin Chem Res15:6052-61). Since this initial work, we showed that a highly stablelinkage of SN-38 was significantly less effective than the CL2A linkerthat has a more intermediate stability in serum (Govindan et al., 2013,Mol Cancer Ther 12:968-78).

Another current tenet of ADC design is to use an ultra-cytotoxic drug tocompensate for low levels of antibody accretion in tumors, typically0.003 to 0.08% of the injected dose per gram (Sharkey et al., 1995,Cancer Res 55:5935s-45s). The current generation of ultratoxic-drugconjugates have found a drug:antibody substitutions of ≤4:1 to beoptimal, since higher ratios adversely affected their pharmacokineticsand diminished the therapeutic index by collateral toxicities (Hamblettet al., 2004, Clin Cancer Res 10:7063-70). In this second-generation ADCplatform, we elected to use an IgG-coupling method thatsite-specifically links the drug to the interchain disulfides throughmild reduction of the IgG, which exposes 8 binding sites. With theCL2A-SN-38 linker, we achieved a DAR of 7.6:1, with LC-MS data showingeach of the 8 coupling sites bears the CL2A linker, but apparently someSN-38 is lost during the manufacturing procedure. Nevertheless, 95% ofthe CL2A linker has 7-8 SN-38 molecules. We found subsequently that (a)coupling to these sites does not destabilize the antibody, and (b)conjugates prepared with these sites substituted at higher levels didnot compromise antibody binding, nor did it affect pharmacokineticproperties. Indeed, we demonstrated that conjugates prepared at themaximum substitution level had the best therapeutic response inmouse-human tumor xenograft models.

One of the more notable features of IMMU-132 from a tolerabilityperspective is that the SN-38 bound to IgG is not glucuronidated, whichis a critical step in the detoxification of irinotecan. With irinotecantherapy, most of the SN-38 generated is readily converted in the liverto the inactive SN-38G form. Estimates of the AUC for SN-38G show it isoften 4.5- to 32-times higher than SN-38 (Gupta et al., 1994, Cancer Res54:3723-25; Xie et al., 2002, J Clin Oncol 20:3293-301). SN-38G'ssecretion into the bile and subsequent deconjugation bybeta-glucuronidase produced by the intestinal flora is stronglyimplicated in the enterohepatic recirculation of SN-38 and the delayedsevere diarrhea observed with irinotecan (Stein et al., 2010, Ther AdvMed Oncol 2:51-63). After IMMU-132 administration, concentrations ofSN-38G were very low in our animal and clinical studies (e.g., in theserum of patients given IMU-132, only 20-40% of the free SN-38 levelsare in the form of SN-38G), providing strong evidence that SN-38 boundto IgG is largely protected from glucuronidation, even though the10-hydroxy position of the SN-38 is available. We speculate that lowlevels of SN-38G generated by IMMU-132 contributes to the lowerincidence and intensity of diarrhea in patients receiving this ADCcompared to irinotecan therapy.

Preventing glucuronidation of the SN-38 bound to the antibody may alsocontribute to improved therapeutic effects for SN-38 delivered to thetumor. Extracts of tumors from animals given irinotecan found highlevels of irinotecan, with 10-fold lower concentrations of SN-38 andSN-38G. In contrast, the only SN-38 found in the tumors of animals givenIMMU-132 was SN-38 bound to the IgG. We hypothesize that the conjugateretained in the tumor will eventually be internalized, thereby releasingits SN-38 payload, or SN-38 could be release outside the tumor cell;however, it would be released in its fully active form, with a lowerlikelihood of being converted to SN-38G, which occurs primarily in theliver. It is also important to emphasize that by coupling the linker tothe 20-hydroxy position of SN-38, the SN-38 is maintained in the activelactone form (Zhao et al., 2000, J Org Chem 65:4601-6). Collectively,these results suggest that IMMU-132 is able to deliver and concentrateSN-38 to Trop-2⁺ tumors in a selective manner compared to SN-3 8 derivedfrom non-targeted irinotecan, with the SN-3 8 delivered by IMMU-132likely being released in the tumor in the fully active,non-glucuronidated, lactone form.

Irinotecan is not conventionally used to treat breast cancer patients.However, the experiments shown here with TNBC cell lines indicate thatconcentrating higher amounts of SN-38 into the tumor enhances itsactivity. In both the MDA-MB-468 TNBC and HER2⁺ SK-BR-3 tumor lines,IMMU-132 mediated the activation of the intrinsic apoptotic pathway,with cleavage of pro-caspases into their active fragments and PARPcleavage. The demonstration of double-stranded DNA breaks of cancercells treated with IMMU-132 (Bardia et al., 2014, San Antonio BreastCancer Symposium, P5-19-27) compared to an irrelevant SN-38 ADC)confirms the selective delivery of SN-38 into the target cells. Mostimportantly, these laboratory findings are confirmed by therapy ofpatients with heavily-pretreated, metastatic TNBC, where durableobjective responses have been observed (Bardia et al., 2014, San AntonioBreast Cancer Symposium, P5-19-27). It also appears that IMMU-132 isactive in patients with other cancers and who have failed a priortherapy regimen containing a topoisomerase I inhibitor (Starodub et al.,2015, Clin Cancer Res 21:3870-78).

In conclusion, the use of SN-38 conjugated at a very high ratio of drugto antibody, using a moderately-stable linker, is efficacious in animalmodels and also clinically, constituting a second-generation ADCplatform. Our findings indicate that Trop-2 is a clinically-relevant andnovel target in Trop-2+ solid tumors, particularly TNBC.

Example 16. Studies on the Mechanism of Action of IMMU-132

Sacituzumab govitecan (IMMU-132, also known as hRS7-CL2A-SN-38) is anantibody-drug conjugate (ADC) targeting Trop-2, a surface glycoproteinexpressed on many epithelial tumors, for delivery of SN-38, the activemetabolite of irinotecan. Unlike most ADCs that use ultratoxic drugs andstable linkers, IMMU-132 uses a moderately toxic drug with a moderatelystable carbonate bond between SN-38 and the linker. Flow cytometry andimmunohistochemistry disclosed Trop-2 is expressed in a wide range oftumor types, including gastric, pancreatic, triple-negative breast(TNBC), colonic, prostate, and lung. While cell-binding experimentsreveal no significant differences between IMMU-132 and parental hRS7antibody, surface plasmon resonance analysis using a Trop-2 CM5 chipshows a significant binding advantage for IMMU-132 over hRS7. Theconjugate retained binding to the neonatal receptor, but lost greaterthan 60% of the antibody-dependent cell-mediated cytotoxicity activitycompared to hRS7.

Exposure of tumor cells to either free SN-38 or IMMU-132 demonstratedthe same signaling pathways, with pJNK1/2 and p21WAF1/Cip1 up-regulationfollowed by cleavage of caspases 9, 7, and 3, ultimately leading topoly-ADP-ribose polymerase cleavage and double-stranded DNA breaks.

Pharmacokinetics of the intact ADC in mice reveals a mean residence time(MRT) of 15.4 h, while the carrier hRS7 antibody cleared at a similarrate as unconjugated antibody (MRT =˜300 h). IMMU-132 treatment of micebearing human gastric cancer xenografts (17.5 mg/kg; twice weekly×4weeks) resulted in significant anti-tumor effects compared to micetreated with a non-specific control. Clinically relevant dosing schemesof IMMU-132 administered either every other week, weekly, or twiceweekly in mice bearing human pancreatic or gastric cancer xenograftsdemonstrate similar, significant anti-tumor effects in both models.Current Phase I/11 clinical trials (ClinicalTrials.gov, NCT01631552)confirm anticancer activity of IMMU-132 in cancers expressing Trop-2,including gastric and pancreatic cancer patients.

Introduction

There will be an estimated 22,220 new cases of gastric cancer diagnosedin the United States this year, with a further 10,990 deaths attributedto this disease (Siegel et al., 2014, CA Cancer J Clin 64:9-29). While5-year survival rates are trending upward (currently at 29%), they arestill quite low when compared to most others, including cancers of thecolon, breast, and prostate (65%, 90%, and 100%, respectively). In fact,among human cancers, only esophageal, liver, lung, and pancreatic haveworse 5-year survival rates. Pancreatic cancer remains the fourthleading cause of all cancer deaths in the U.S., with a 5-year survivalrate of only 6% (Siegel et al., 2014, CA Cancer J Clin 64:9-29). It isclear from such grim statistics for gastric and pancreatic cancer thatnew therapeutic approaches are needed.

Trop-2 is a 45-kDa glycoprotein that belongs to the TACSTD gene family,specifically TACSTD22. Overexpression of this trans-membrane protein onmany different epithelial cancers has been linked to an overall poorprognosis. Trop-2 is essential for anchorage-independent cell growth andtumorigenesis (Wang et al., 2008, Mol Cancer Ther 7:280-85; Trerotola etal., 2013, Oncogene 32:222-33). It functions as a calcium signaltransducer that requires an intact cytoplasmic tail that isphosphorylated by protein kinase C12-14. Pro-growth signaling associatedwith Trop-2 includes NF-κB, cyclin D1 and ERK (Guerra et al., 2013,Oncogene 32:1594-1600; Cubas et al., 2010, Mol Cancer 9:253).

In pancreatic cancer, Trop-2 overexpression was observed in 55% ofpatients studied, with a positive correlation with metastasis, tumorgrade, and poor progression-free survival of patients who underwentsurgery with curative intent (Fong et al., 2008, Br J Cancer99:1290-95). Likewise, in gastric cancer 56% of patients exhibitedTrop-2 overexpression on their tumors, which again correlated withshorter disease-free survival and a poorer prognosis in those patientswith lymph node involvement of Trop-2-positive tumor cells (Muhlmann etal., 2009, J Clin Pathol 63:152-58). Given these characteristics and thefact that Trop-2 is linked to so many intractable cancers, Trop-2 is anattractive target for therapeutic intervention with an antibody-drugconjugate (ADC).

A general paradigm for using an antibody to target a drug to a tumorincludes several key features, among them: (a) an antigen target that ispreferentially expressed on the tumor versus normal tissue, (b) anantibody that has good affinity and is internalized by the tumor cell,and (c) an ultra-toxic drug that is coupled stably to the antibody(Panowski et al., 2014, mAbs 6:34-45). Along these lines, we developedan antibody, designated RS7-3G11 (RS7), that bound to Trop-2 in a numberof solid tumors (Stein et al., 1993, Int J Cancer 55:938-46; Basu etal., 1995, Int J Cancer 62:472-79) with nanomolar affinity (Cardillo etal., 2011k, Clin Cancer Res 17:3157-69), and once bound to Trop-2, isinternalized by the cell (Shih et al., 1995, Cancer Res 55:5857s-63s).

By immunohistochemistry, Trop-2 is expressed in some normal tissues,though usually at much lower intensities when compared to neoplastictissue, and often is present in regions of the tissues with restrictedvascular access (Trerotola et al., 2013, Oncogene 32:222-33). Based onthese characteristics, RS7 was humanized and conjugated with the activemetabolite of irinotecan, 7-ethyl-10-hydroxycamptothecin (SN-38). Invitro cytotoxicity in numerous cell lines has found IC₅₀-values in thesingle digit nanomolar range for SN-38, compared to picomolar range formany of the ultra-toxic drugs currently used in ADCs (Cardillo et al.,2011, Clin Cancer Res 17:3157-69). While the prevailing opinion is touse ultra-toxic agents, such as auristatins or maytansines, to make ADCswith only 2-4 drugs per antibody linked stably to the antibody, suchagents have a narrow therapeutic window, resulting in renewed efforts tore-engineer ADCs to broaden their therapeutic index (Junutula et al.,2010, Clin Cancer Res 16:4769-78).

As one approach to diverge from this practice, we conjugated 7-8 SN-38molecules per antibody using a linker that releases SN-38 with half-lifeof ˜1 day in human serum. It is hypothesized that using a less stablelinker allows for SN-38 release at the tumor site after the ADC targetsthe cells, making the drug accessible to surrounding tumor cells and notjust cells directly targeted by the ADC. The resulting ADC,hRS7-CL2A-SN-38 (sacituzumab govitecan, or IMMU-132), has shownanti-tumor activity against a wide range of tumor types (Cardillo etal., 2011, Clin Cancer Res 17:3157-69). More recently, IMMU-132 hasdemonstrated significant anti-tumor activity against a pre-clinicalmodel of triple negative breast cancer (TNBC) (Goldenberg et al., 2014,Poster presented at San Antonia Breast Cancer Symposium, December 9-13,Abstr. P5-19-08). Most importantly, in a current Phase I/II clinicaltrial, IMMU-132 has shown activity in TNBC patients (Bardia et al.,2014, Poster presented at San Antonia Breast Cancer Symposium, December9-13, Abstr. P5-19-2), thus validating this paradigm shift in ADCchemistry using a less toxic drug and a linker that releases SN-38 overtime rather than being totally dependent on internalization of the ADCto achieve activity.

SN-38 is a known topoisomerase-I inhibitor that induces significantdamage to a cell's DNA. It mediates the up-regulation of earlypro-apoptotic proteins, p53 and p21WAF1/Cip1, resulting in caspaseactivation and poly-ADP-ribose polymerase (PARP) cleavage. Expression ofp21WAF1/Cip1 is associated with G1 arrest of the cell cycle and is thusa hallmark of the intrinsic apoptotic pathway. We demonstratedpreviously that IMMU-132 likewise could mediate the up-regulation ofearly pro-apoptosis signaling events (p53 and p21WAF1/Cip1) resulting inPARP cleavage in NSCLC (Calu-3) and pancreatic (BxPC-3) cell linesconsistent with the intrinsic pro-apoptosis signaling pathway (Cardilloet al., 2011, Clin Cancer Res 17:3157-69).

Herein, we further characterize IMMU-132, with particular attentiontowards the treatment of solid cancers, especially human gastric andpancreatic tumors. Trop-2 surface expression across a range of solidtumor types is examined and correlated with in vivo expression in tumorxenografts. Mechanistic studies further elucidate the intrinsicpro-apoptotic signaling events mediated by IMMU-132, including evidenceof increased double stranded DNA (dsDNA) breaks and later caspaseactivation. Finally, clinically-relevant and non-toxic dosing schemesare compared in gastric and pancreatic carcinoma disease models, testingtwice-weekly, weekly, and every other week schedules to ascertain whichtreatment cycle may be best applied to a clinical setting without lossof efficacy.

Experimental Procedures

Cell Lines and Chemotherapeutics—All human cancer cell lines used werepurchased from the American Type Culture Collection (ATCC) (Manassas,Va.). Each was maintained according to the recommendations of ATCC androutinely tested for mycoplasma, and all were authenticated by shorttandem repeat (STR) assay by the ATCC. IMMU-132 (hRS7-SN-38) and controlADCs (anti-CD20 hA20-SN-38 and anti-CD22 hLL2-SN-38) were made aspreviously described and stored at −20° C. (Cardillo et al., 2011, ClinCancer Res 17:3157-69). SN-38 was purchased (Biddle Sawyer Pharma, LLC,New York, N.Y.) and stored in 1 mM aliquots in DMSO at −20° C.

Trop-2 ELISA—Recombinant human Trop-2 with a His-tag (Sino Biological,Inc., Bejing, China; Cat# 10428-H09H) and recombinant mouse Trop-2 witha His-Tag (Sino Biological, Inc., Cat# 50922-M08H) were plated ontoNi-NTA Hissorb strips (Qiagen GmbH Cat# 35023) at 1 μg for 1 h at roomtemperature. The plate was washed four times with PBS-Tween (0.05%) washbuffer. Serial dilutions of hRS7 were made in 1% BSA-PBS dilution bufferto a test range of 0.1 ng/mL to 10 μg/mL. The plates were then incubatedfor 2 h at room temperature before being washed four times followed bythe addition of a peroxidase conjugated secondary antibody (goatanti-human, Fc fragment specific; Jackson Immunoresearch Cat#109-036-098). After a 45-min incubation, the plate was washed and asubstrate solution (o-phenylenediamine dihydrochloride (OPD); Sigma,Cat#P828) added to all the wells. Plates were incubated in the dark for15 min before the reaction was stopped with 4N sulfuric acid. The plateswere read at 450 nm on Biotek ELX808 plate reader. Data were analyzedand graphed using Prism GraphPad Software (v4.03) (Advanced GraphicsSoftware, Inc.; Encinitas, Calif.).

In Vitro Cell Binding—LumiGLO Chemiluminescent Substrate System (KPL,Gaithersberg, Md.) was used to detect antibody binding to cells.Briefly, cells were plated into a 96 black-well, flat-clear-bottom plateovernight. Antibodies were serially diluted 1:2 and added in triplicate,yielding a concentration range from 0.03 to 66.7 nM. After incubatingfor 1 h at 4° C., the media was removed and the cells washed with freshcold media followed by the addition of a 1:20,000 dilution ofgoat-antihuman horseradish peroxidase-conjugated secondary antibody(Jackson Immunoresearch, West Grove, Pa.) for 1 h at 4° C. The plateswere again washed before the addition of the LumiGLO reagent. Plateswere read for luminescence using an Envision plate reader (Perkin Elmer,Boston Mass.). Data were analyzed by non-linear regression to determinethe equilibrium dissociation constant (KD). Statistical comparisons ofKD -values were made with Prism GraphPad Software (v4.03) (AdvancedGraphics Software, Inc.; Encinitas, Calif.) using an F-Test on thebest-fit curves for the data. Significance was set at P<0.05.

Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC)—A four-hourLDH-release assay was performed to evaluate ADCC activity elicited byIMMU-132, hRS7 IgG, hLL2-SN-38 and hLL2 IgG (hLL2 are non-bindinganti-CD22 conjugates for the solid tumor cell lines). Briefly, targetcells (MDA-MB-468, NIH:OVCAR-3, or BxPC-3) were plated at 1×10⁴cells/well in a 96-well, black, flat-bottom plate and incubatedovernight. The next day, peripheral blood mononuclear effector cells(PBMCs) were freshly isolated from a donor and added to assigned wellson the reaction plate at an E:T ratio of 50:1. Acquisition of humanPBMCs was done under the approval of the New England InstitutionalReview Board (Newton, Mass.). Test reagents were added to their assignedwells at a final concentration of 33.3 nM. One set of wells receivedADCC assay medium alone for background control and another set of wellsreceived cells alone plus TritonX100 for maximum cells lysis control.The plate was incubated for 4 h at 37° C. After 4 h, target cell lysiswas assessed by a homogenous fluorometric LDH release assay (CytoTox-One Homogenous Membrane Integrity Assay; Promega, Cat. G7891).

The plates were read (544 nm-590 nm) using an Envision plate reader(PerkinElmer LAS, Inc.; Shelton, Conn.). Data were analyzed by MicrosoftExcel. Percent specific lysis was calculated as follows:

${\% \mspace{14mu} {Specific}\mspace{14mu} {Lysis}} = {\frac{{Experimental} - \left( {{Effector} + {{Target}\mspace{14mu} {Control}}} \right)}{{{Max}.\mspace{14mu} {Lysis}} - \left( {{Target}\mspace{14mu} {Control}} \right)} \times 100}$

where:

Experimental: effector+target cells+antibody

Effector+Target Control: effector+target cells

Max. Lysis: target cells+Triton-X100

Target Control: target cells only

Surface Plasmon Resonance Binding (BIACORE)—Briefly, rhTrop-2/TACSTD2(Sino Biological, Inc.) or recombinant human neonatal receptor (FcRn),produced as described (Wang et al., 2011, Drug Metab Dispos 39:1469-77),was immobilized with an amine coupling kit (GE Healthcare; Cat. No.BR-1000-50) on a CM5 sensor chip (GE Healthcare; Cat. #BR-1000-12)following manufacturer's instructions for a low-density chip. Threeseparate sets of dilutions of hRS7 IgG and IMMU-132 were made in runningbuffer (400 nM, 200 nM, 100 nM, 50 nM and 25 nM). Each set would make upa separate run on the BIACORE (BIACORE-X; Biacore Inc., Piscataway,N.J.) and data were analyzed using BIAevaluation Software (Biacore Inc.,v4.1). Analysis was performed with a 1:1 (Langmuir) Binding Model andFit, using all five concentration points for each sample run todetermine the best fit (lowest χ² value). The KDvalue was calculatedusing the formula KD=kd1/ka1, where kd1 is the dissociationrate-constant and ka1 is the association-rate constant.

Immunohistological Assessment of the Distribution of Trop-2 inFormalin-Fixed, Paraffin-Embedded Tissues—Tumor xenografts were takenfrom mice, fixed in 10% buffered formalin and paraffin-embedded. Afterde-paraffination, 5 μm sections were incubated with Tris/EDTA buffer(DaKo Target Retrieval Solution, pH 9.0; Dako, Denmark), at 95° C. for30 min in a NxGen Decloaking Chamber (Biocare Medical, Concord, Calif.).Trop-2 was detected with a goat polyclonal antihuman Trop-2 antibody at10 μg/mL (R&D Systems, Minneapolis, Minn.) and stained with VectorVECTASTAINR ABC Kit (Vector Laboratories, Inc., Burlingame, Calif.).Normal goat antibody was used as the negative control (R&D Systems,Minneapolis, Minn.). Tissues were counterstained with hematoxylin for 6seconds.

Trop-2 Surface Expression on Human Carcinoma Cell Lines—Expression ofTrop-2 on the cell surface is based on flow cytometry. Briefly, cellswere harvested with Accutase Cell Detachment Solution (Becton Dickinson(BD), Franklin Lakes, N.J.; Cat. No. 561527) and assayed for Trop-2expression using QuantiBRITE PE beads (BD Cat. No. 340495) and aPE-conjugated anti-Trop-2 antibody (eBiosciences, Cat. No. 12-6024)following the manufactures' instructions. Data were acquired on aFACSCalibur Flow Cytometer (BD) with CellQuest Pro software. Stainingwas analyzed with Flowjo software (Tree Star, Ashland Oreg.).

Pharmacokinetics—Naive female NCr nude (nu/nu) mice, 8-10 weeks old,were purchased from Taconic Farms (Germantown, N.Y.). Mice (N=5) wereinjected i.v. with 200 μg of IMMU-132, parental hRS7, or modifiedhRS7-NEM (hRS7 treated with TCEP and conjugated with N-ethylmaleimide).Animals were bled via retroorbital plexis at 30-min, 4-, 24-, 72- and168-h post-injection. ELISA was utilized to determine serumconcentrations of total hRS7 IgG by competing for the binding to ananti-hRS7 IgG idiotype antibody with a horseradish peroxidase conjugateof hRS7. Serum concentrations of intact IMMU-132 were determined usingan anti-SN-38 antibody to capture and a horseradishperoxidase-conjugated anti-hRS7 IgG antibody to detect. Pharmacokinetic(PK) parameters were computed by noncompartmental analysis using PhoenixWinNonlin software (version 6.3; Pharsight Corp., Mountainview, Calif.).

Assessment of Double-Stranded DNA Breaks In Vitro—For drug activitytesting, BxPC-3 cells were seeded in 6-well plates at 5×105 cells/welland held at 37° C. overnight. After 10 min cooling on ice, cells wereincubated with IMMU-132, hA20-SN-38, or hRS7-IgG at the finalconcentration of 20 μg/ml for 30 min on ice, washed three times withfresh media, and then returned to 37° C. to continue culture overnight.The following morning, cells were trypsinized briefly, spun down,stained with Fixable Viability Stain 450 (BD Biosciences, San Jose,Calif.), washed with 1% BSA-PBS, and then fixed in 4% formalin for 15min, washed again and permeabilized in 0.15% Triton-X100 in PBS foranother 15 min. After washing twice with 1% BSA-PBS, cells wereincubated with mouse anti-yH2AX-AF488 (EMD Millipore Corporation,Temecula, Calif.) for 45 min at 4° C. The signal intensity of yH2AX wasmeasured by flow cytometry using a BD FACSCanto (BD Biosciences, SanJose, Calif.).

In Vivo Therapeutic Studies—NCr female athymic nude (nu/nu) mice, 4-8weeks old, were purchased from Taconic Farms (Germantown, N.Y.). NCI-N87gastric tumor xenografts were established by harvesting cells fromtissue culture and making a final cell suspension 1:1 in matrigel (BDBioscience; San Jose, Calif.), with each mouse receiving a total of1×10⁷ cells s.c. in the right flank. For BxPC-3. xenografts of 1 g wereharvested, and a tumor suspension made in HBSS to a concentration of 40%tumor w/v. This suspension was mixed 1:1 with matrigel for a final tumorsuspension of 20% w/v. Mice were then injected with 300 μL s.c. Tumorvolume (TV) was determined by measurements in two dimensions usingcalipers, with volumes defined as: L×w²/2, where L is the longestdimension of the tumor and w the shortest. For IHC, tumors were allowedto grow to approximately 0.5 cm³ before the mice were euthanized and thetumors removed, formalin-fixed and paraffin-embedded. For therapystudies, mice were randomized into treatment groups and therapy begunwhen tumor volumes were approximately 0.25 cm³. Treatment regimens,dosages, and number of animals in each experiment are described in theResults and in the Figure legends. The lyophilized IMMU-132 and controlADC (hA20-SN-38) were reconstituted and diluted as required in sterilesaline.

Mice were euthanized and deemed to have succumbed to disease once tumorsgrew to greater than 1.0 cm³ in size. Best responses to therapy weredefined as: partial response, shrinking >30% from starting size; stabledisease, tumor volumes shrinking up to 29% or increase no greater than20% of initial size; progression, tumors increase ≥20% either from theirstarting size or from their nadir. Time to progression (TTP) wasdetermined as time post-therapy initiation when the tumor grew more than20% in size from its nadir.

Statistical analysis of tumor growth was based on area under the curve(AUC). Profiles of individual tumor growth were obtained throughlinear-curve modeling. Anf-test was employed to determine equality ofvariance between groups prior to statistical analysis of growth curves.A two-tailed t-test was used to assess statistical significance betweenthe various treatment groups and controls, except for the salinecontrol, where a one-tailed t-test was used (significance at P≤0.05).Survival studies were analyzed using Kaplan-Meier plots (log-rankanalysis), using the Prism GraphPad Software (v4.03) software package(Advanced Graphics Software, Inc., Encinitas, Calif.).

Immunoblotting—Cells (2×10⁶) were plated in 6-well plates overnight. Thefollowing day they were treated with either free SN-38 (dissolved inDMSO) or IMMU-132 at an SN-38 concentration equivalent to 0.4 μg/mL (1μM). Parental hRS7 was used as a control for the ADC. Cells were lysedin buffer containing 10 mM Tris, pH 7.4, 150 mM NaCl, proteaseinhibitors and phosphatase inhibitors (2 mM Na2PO4, 10 mM NaF). A totalof 20 μg protein was resolved in a 4-20% SDS polyacrylamide gel,transferred onto a nitrocellulose membrane and blocked by 5% non-fatmilk in 1×TBS-T (Tris-buffered saline, 0.1% Tween-20) for 1 h at roomtemperature. Membranes were probed overnight at 4° C. with primaryantibodies followed by 1-h incubation with antirabbit secondary antibody(1:2500) at room temperature. Signal detection was done using achemiluminescence kit (Supersignal West Dura, Thermo Scientific;Rockford, Ill.) with the membranes visualized on a Kodak Image Station40000R. Primary antibodies p21Waf1/Cip1 (Cat. No. 2947), Caspase-3 (Cat.No. 9665), Caspase-7 (Cat. No. 9492), Caspase-9 (Cat. No. 9502), PARP(Cat. No. 9542), β-actin (Cat. No. 4967), pJNK1/2 (Cat. No. 4668), JNK(Cat. No. 9258), and goat anti-rabbit-HRP secondary antibody (Cat. No.7074) were obtained from Cell Signal Technology (Danvers, Mass.).

Results

Trop-2 Expression Levels in Multiple Solid Tumor Cell Lines—Surfaceexpression of Trop-2 is evident in a variety of human solid tumor lines,including gastric, pancreatic, breast, colon, and lung (Table 14). Thereis no one tumor type that had higher expression above any other, withvariability observed within a given tumor cell type. For example, withingastric adenocarcinomas, Trop-2 levels ranged from very low 494±19 (Hs746T) to high 246,857±64,651 (NCI-N87) surface molecules per cell.

Gastrointestinal tumor xenografts stained for Trop-2 expression showedboth cytoplasmic and membrane staining (not shown). Staining intensitycorrelated well with the results for surface Trop-2 expressiondetermined by FACS analysis. For the pancreatic adenocarcinomas, allthree had homogenous staining, with BxPC-3 representing 2+ to 3+staining. NCI-N87 gastric adenocarcinoma had a more heterogeneousstaining pattern, with 3+ staining of the apical lining of the glandsand less pronounced staining of surrounding tumor cells. COLO 205demonstrated only very focal 1+ to 2+ staining, whereas HT-29 showedvery rare 1+ staining of a few cells.

TABLE 14 Trop-2 surface expression levels in various solid tumor linesvia FACS analysis. a Number of Surface Trop-2 Molecules per Cell CellLine Mean ± SD Gastric NCI-N87 246,857 ± 64,651  AGS 53,756 ± 23,527 Hs746T 494 ± 19  Pancreatic BxPC-3 493,773 ± 97,779  CFPAC-1 162,871 ±28,161  Capan-1 157,376 ± 36,976  HPAF-II 115,533 ± 28,627  Breast (TN)MDA-MB-468 301,603 ± 29,470  HCC38 181,488 ± 69,351  HCC 1806 91,403 ±20,817 MDA-MB-231 32,380 ± 5,460  Breast SK-BR-3 (HER2+) 328,281 ±47,996  MCF-7 (ER2+) 110,646 ± 17,233  Colon COLO 205 58,179 ± 6,909HT-29 68 ± 17 NSCLC Calu-3 128,201 ± 50,708  Sq. Cell Lung SK-MES-129,488 ± 5,824  Acute T-Cell Leukemia Jurkat 0 a Three separate assayswere performed, with the mean and standard deviation provided.

IMMU-132 Binding Characteristics—To further demonstrate that hRS7 doesnot cross-react with murine Trop-2, an ELISA was performed on platescoated with either recombinant murine Trop-2 or human Trop-2 (notshown). Humanized RS7 specifically bound only to the human Trop-2(KD=0.3 nM); there was no cross-reactivity with the murine Trop-2.Control polyclonal rabbit anti-murine Trop-2 and antihuman Trop-2antibodies did cross-react and bound to both forms of Trop-2 (data notshown).

IMMU-132 binding to multiple cell lines was examined, with comparison toparental hRS7 as well as to modified hRS7, hRS7-NEM (hRS7 treated withTCEP and conjugated with N-ethylmaleimide) (not shown). In all cases,calculated KD-values were in the sub-nanomolar range, with nosignificant differences between hRS7, IMMU-132, and hRS7-NEM within agiven cell line.

Comparisons in binding of IMMU-132 and hRS7 were further investigatedusing surface plasmon resonance (BIACORE) analysis (not shown). Alow-density Trop-2 biosensor chip (density=1110 RU) was utilized withrecombinant human Trop-2. Not only did three independent binding runsdemonstrate that IMMU-132 is not affected adversely by theSN-38-conjugation process, but it demonstrated a higher binding affinityto Trop-2 than hRS7 (0.26±0.14 nM vs. 0.51±0.04 nM, respectively;P=0.0398).

Mechanism of Action: ADCC and Intrinsic Apoptosis SignalingPathways—ADCC activity of IMMU-132 was compared to hRS7 in threedifferent cell lines, TNBC (MDAMB-468), ovarian (NIH:OVCAR-3), andpancreatic (BxPC-3) (FIG. 21). In all three, hRS7 significantly mediatedcell lysis compared to all other treatments, including IMMU-132(P<0.0054). ADCC decreased by more than 60% when IMMU-132 was used totarget the cells as compared to hRS7. For example, in MDA-MB-468,specific lysis mediated by hRS7 was 29.8±2.6% versus 8.6±2.6% forIMMU-132 (FIG. 21 (A); P<0.0001). Similar loss in ADCC activity waslikewise observed in NIH:OVCAR-3 and BxPC-3 (FIG. 21 (B) and FIG. 21(C); P<0.0001 and P<0.0054; respectively). This diminished ADCC activityappears to be the result of changes to the antibody during theconjugation process, since this same loss in specific cell lysis wasevident with hRS7-NEM, which lacks the CL2A-SN-38 linker, having thecysteines blocked instead with N-ethylmaleimide (FIG. 21 (C)). There isno CDC activity associated with hRS7 or IMMU-132 (data not shown).

IMMU-132 has been shown previously to mediate the up-regulation of earlypro-apoptosis signaling events (p53 and p21WAF1/Cip1), ultimatelyleading to the cleavage of PARP20. In order to better define theapoptotic pathway utilized by IMMU-132, the NCI-N87 human gastriccarcinoma and BxPC-3 pancreatic adenocarcinoma cell lines were exposedto 1 μM of free SN-38 or the equivalent amount of IMMU-132 (not shown).Both free SN-38 and IMMU-132 mediate the up-regulation of p21WAF1/Cip1,though it is not until 48 h that the up-regulation between the NCI-N87cells exposed to free SN-38 versus IMMU-132 are the same (not shown),whereas in BxPC-3 maximum up-regulation is evident within 24 h (notshown). Both free SN-38 and IMMU-132 demonstrate cleavage ofpro-caspase-9 and -7 within 48 h of exposure. Procaspase-3 is cleaved inboth cell lines with the highest degree of cleavage observed after 48 h.Finally, both free SN-38 and IMMU-132 mediated PARP cleavage. This firstbecomes evident at 24 h, with increased cleavage at 48 h. Takentogether, these data confirm that the SN-38 contained in IMMU-132 hasthe same activity as free SN-38.

In addition to these later apoptosis signaling events, an earlier eventassociated with this pathway, namely the phosphorylation of JNK (pJNK),is also evident in BxPC-3 cells exposed for a short time to either freeSN-38 or IMMU-132, but not naked hRS7 (not shown). Increased amounts ofpJNK are evident by 4 h, with no appreciable change at 6 h. There is ahigher intensity of phosphorylation in the cells exposed to free-SN-38as compared to IMMU-132, but both are substantially higher thancontrols. As an end-point for the mechanism of action of IMMU-132,measurements of dsDNA breaks were made in BxPC-3 cells. Exposure ofBxPC-3 to IMMU-132 for only 30 min resulted in a greater than two-foldinduction of γH2AX when compared to a non-targeting control ADC (Table15). Approximately 70% of the cells were positive for γH2AX stainingversus <20% for naked hRS7, hA20-SN-38 irrelevant ADC, and untreatedcontrols (P<0.0002).

TABLE 15 IMMU-132-mediated dsDNA breaks in BxPC-3: γH2AX induction.^(a)Treatment Mean Fluorescence Intensity Percent Positive Untreated 2516 ±191 18.8 ± 6.3 hRS7 2297 ± 18  13.0 ± 0.6 hA20-SN-38 2246 ± 58  12.7 ±2.4 IMMU-132 5349 ± 234 69.0 ± 4.1 ^(a)IMMU-132 vs. all 3 controltreatments, P < 0.0002 (onetailed t-Test; N = 3).

Pharmacokinetics of IMMU-132—Binding to the human neonatal receptor(FcRn) was determined by BIACORE analysis (not shown). Using alow-density FcRn biosensor chip (density=1302 RU), three independentbinding runs at five different concentrations (400 to 25 nM) wereconducted for each agent. Overall, both hRS7 and IMMU-132 demonstrateKD-values in the nanomolar range (92.4±5.7 nM and 191.9±47.6 nM,respectively), with no significant difference between the two. Mice wereinjected with IMMU-132, with the clearance of IMMU-132 versus the hRS7IgG compared to the parental hRS7 using two ELISAs (FIG. 22). Miceinjected with hRS7 demonstrated a biphasic clearance pattern (FIG. 22(A)) that was similar to what was observed for the hRS7 targetingportion of IMMU-132 (FIG. 22 (B)), with alpha and beta half-lives ofapproximately 3 and 200 h, respectively. In contrast, a rapid clearanceof intact IMMU-132 was observed with a half-life of 11 h and meanresidence time (MRT) of 15.4 h (FIG. 22 (C)).

To further confirm that disruption of interchain disulfide bonds doesnot alter the PK of the targeting antibody, the PK of parental hRS7 wascompared to modified hRS7 (hRS7-NEM). There were no significantdifferences noted between either agent in terms of half-life, Cmax, AUC,clearance, or MRT (not shown).

IMMU-132 Efficacy in Human Gastric Carcinoma Xenografts—Efficacy ofIMMU-132 has been demonstrated previously in non-small-cell lung, colon,TNBC, and pancreatic carcinoma xenograft models (Cardillo et al., 2011,Clin Cancer Res 17:3157-69; Goldenberg et al., Poster presented at SanAntonio Breast Cancer Symposium, December 9-13, Abstr P5-19-08). Tofurther extend these findings to other gastrointestinal cancers,IMMU-132 was tested in mice bearing a human gastric carcinoma xenograft,NCI-N87 (FIG. 23). Treatment with IMMU-132 achieved significant tumorregressions compared to saline and non-targeting hA20 (anti-CD20)-SN-38ADC controls (FIG. 23 (A); P<0.001). There were 6 of 7 mice in theIMMU-132 group that were partial responders that lasted for more than 18days after the last therapy dose was administered to the animals. Thisresulted in a mean time to progression (TTP) of 41.7±4.2 days comparedto no responders in the control ADC group, with a TTP of 4.1±2.0 days(P<0.0001). Overall, the median survival time (MST) for IMMU-132-treatedmice was 66 days versus 24 days for control ADC and 14 days for salinecontrol animals (FIG. 23 (B); P<0.0001).

Clinically-Relevant Dosing Schemes—The highest repeated doses toleratedof IMMU-132 currently being tested clinically are 8 and 10 mg/kg givenon days 1 and 8 of 21-day cycles. A human dose of 8 mg/kg translates toa murine dose of 98.4 mg/kg, or approximately 2 mg to a 20 g mouse.Three different dose schedules of fractionated 2 mg of IMMU-132 wereexamined in a human pancreatic adenocarcinoma xenograft model (BxPC-3).This total dose was fractionated using one of three different dosingschedules: one group received two IMMU-132 doses of 1 mg (therapy days 1and 15), one received four doses of 0.5 mg (therapy days 1, 8, 22, and29), and the final group eight doses of 0.25 mg (therapy days 1, 4, 8,11, 22, 25, 29, and 32). All three dosing schemes provided a significantanti-tumor effect when compared to untreated control animals, both interms of tumor growth inhibition and overall survival (FIG. 24 (A);P<0.0009 and P<0.0001, respectively). There are no significantdifferences in TTP between the three different treatment groups, whichranged from 22.4±10.1 days for the 1-mg dosing group to 31.7±14.5 daysfor the 0.25-mg dosing group (TTP for untreated control group=5.0±2.3days).

A similar dose schedule experiment was performed in mice bearing NCI-N87human gastric tumor xenografts (FIG. 24 (B)). All three dose scheduleshad a significant anti-tumor effect when compared to untreated controlmice but were no different from each other (AUC; P<0.0001). Likewise, interms of overall survival, while all three dose schedules provided asignificant survival benefit when compared to untreated control(P<0.0001), there were no differences between any of these threedifferent schedules.

To further discriminate possible dosing schemes, mice bearing NCI-N87tumors were subjected to chronic IMMU-132 dosing in which mice received0.5 mg injections of IMMU-132 once a week for two weeks followed by oneweek off before starting another cycle (FIG. 24 (C)), as in the currentclinical trial schedule. In all, four treatment cycles were administeredto the animals.

This dosing schedule slowed tumor growth with a TTP of 15.7±11.1 daysversus 4.7±2.2 days for control ADC-treated mice (P=0.0122). Overall,chronic dosing increased the median 19 survival 3-fold from 21 days forcontrol ADC-treated mice to 63 days for those animals administeredIMMU-132 (P=0.0001). Importantly, in all these different dosing schemeevaluations, no treatment-related toxicities were observed in the miceas demonstrated by no significant loss in body weight (data not shown).

Discussion

In a current Phase I/II clinical trial (ClinicalTrials.gov,NCT01631552), IMMU-132 (sacituzumab govitecan) is demonstratingobjective responses in patients presenting with a wide range of solidtumors (Starodub et al., 2015, Clin Cancer Res 21:3870-78). As thisPhase I/II clinical trial continues, efficacy of IMMU-132 needs to befurther explored in an expanding list of Trop-2-positive cancers.Additionally, the uniqueness of IMMU-132, in contrast to otherclinically relevant ADCs that make use of ultratoxic drugs, needs to befurther elucidated as we move forward in its clinical development.

The work presented here further characterizes IMMU-132 and demonstratesits efficacy against gastric and pancreatic adenocarcinoma atclinically-relevant dosing schemes. The prevailing view of a successfulADC is that it should use an antibody recognizing an antigen with hightumor expression levels relative to normal tissue and one thatpreferably internalizes when bound to the tumor cells (Panowski et al.,2014, mAbs 6:34-45). All of the currently approved ADCs have used anultra-toxic drug (pM IC50) coupled to the antibody by a highly stablelinker at low substitution ratios (2-4 drugs per antibody). IMMU-132diverges from this paradigm in three main aspects: (i) SN-38, amoderately cytotoxic drug (nM IC₅₀), is used as the chemotherapeuticagent, (ii) SN-38 is conjugated site-specifically to 8 interchain thiolsof the antibody, yielding a substitution of 7.6 drugs per antibody, and(iii) a carbonate linker is used that is cleavable at low pH, but willalso release the drug with a half-life in serum of ˜24 h (Cardillo etal., 2011, Clin Cancer Res 17:3157-69). IMMU-132 is composed of anantibody that internalizes after binding to an epitope, as we haveshown, that is specific for human Trop-2, which is highly expressed onmany different types of epithelial tumors, as well as at lowerconcentrations in their corresponding normal tissues (Shih et al., 1995,Cancer Res 55:5857s-63s). Despite the presence in normal tissues, priorstudies in monkeys, which also express Trop-2 in similar tissues,indicated relatively mild and reversible histopathological changes evenat very high doses where dose-limiting neutropenia and diarrheaoccurred, suggesting the antigen in the normal tissues was sequesteredin some manner, or that the use of a less toxic drug spared these normaltissues from severe damage (Cardillo et al., 2011, Clin Cancer Res17:3157-69).

Herein, we expanded an assessment of Trop-2 expression on multiple humansolid tumor lines, examining in vitro expression in a more quantitativemanner than reported previously, but also, importantly, in xenograftsthat illustrate Trop-2 expression ranging from homogenous (e.g., NCIN87)to very focal (e.g., COLO 205). Overall, surface expression levels ofTrop-2 determined in vitro correlated with staining intensity upon IHCanalysis of xenografts. It is particularly interesting that even in atumor like COLO 205, where there are only focal pockets ofTrop-2-expressing cells revealed by immunohistology, IMMU-132 was stillcapable of eliciting specific tumor regressions, suggesting that abystander effect may occur as a result of the release of SN-38 from theconjugate bound to the antigen-presenting cells (Cardillo et al., 2011,Clin Cancer Res 17:3157-69). Indeed, SN-38 readily penetrates cellmembranes, and therefore its local release within the tumormicroenvironment provides another mechanism for its entry into cellswithout requiring internalization of the intact conjugate. Importantly,the SN-38 bound to the conjugate remains in a fully active state;namely, it is not glucuronidated and would be in the lactone ring format the time of release (Sharkey et al., 2015, Clin Cancer Res,21:5131-8). This property is unique, distinguishing IMMU-132's abilityto localize a fully active form of SN-38 in a more selective manner thanany of the other slow-release SN-38 or irinotecan agents studied todate.

The Phase I clinical trial with IMMU-132 identified 8 to 10 mg/kg givenweekly for two weeks on a 21-day cycle for further investigation inPhase II (Starodub et al., 2015, Clin Cancer Res 21:3870-78). Patientswith a wide range of metastatic solid tumors, including pancreatic andgastric cancers, have shown extended periods of disease stabilizationafter relapsing to multiple prior therapies (Starodub et al., 2015, ClinCancer Res 21:3870-78; Starodub et al., 2014, J Clin Oncol 32:5s (SupplAbstr 3032)). Additional studies in xenograft models were undertaken todetermine if different dosing schedules may be more efficacious. To thisend, the equivalent to the human dose of 8 mg/kg (mouse dose of 98.4mg/kg) was fractionated over three different dosing schedules, includingevery other week, weekly, or twice-weekly on a 21-day cycle. In thepancreatic and gastric tumor models, no significant difference intherapeutic responses were observed for all three schedules, with tumorsprogressing only after therapy was discontinued. Therefore, these datasupport the continued use of the once-weekly dosing regimen currentlybeing pursued clinically.

With clinical trials recommending an IMMU-132 each treatment dose of 8to 10 mg/kg (Starodub et al., 2015, Clin Cancer Res 21:3870-78), it wasimportant to examine whether the antibody alone might contribute to theIMMU-132's activity. Previous studies in nude mice-human xenograftmodels had included unconjugated hRS7 IgG alone (e.g., repeated doses of25 to 50 mg/kg), with no evidence of therapeutic activity (Cardillo etal., 2011, Clin Cancer Res 17:3157-69); however, studies in mice cannotalways predict immunological functionality. ADCC activity of hRS7 invitro has been reported in Trop-2-positive ovarian and uterinecarcinomas (Raji et al., 2011, J Exp Clin Cancer Res 30:106; Bignotti etal., 2011, Int J Gynecol Cancer 21:1613-21; Varughese et al., 2011, Am JObstet Gynecol 205:567; Varughese et al., 2011, Cancer 117:3163-72). Weconfirmed unconjugated hRS7 ADCC activity in three different cellslines, but found IMMU-132 lost 60-70% of its effector function. Sincethe reduced/NEM-blocked IgG has a similar loss of ADCC activity, itappears that the attachment of the CL2A-SN-38 component was not, initself, responsible.

Antibodies also can elicit cell death by acting on various apoptoticsignaling pathways. However, we did not observe any effects of theunconjugated antibody in a number of apoptotic signaling pathways, butinstead noted IMMU-132 elicited similar intrinsic apoptotic events asSN-38. Early events include the phosphorylation of JNK1/2 as well as theup-regulation of p21WAF1/Cip1 leading to the activation of caspase-9,-7, and -3, with the end result of PARP cleavage and significant levelsof dsDNA breaks, as measured by increased amounts of phosphorylatedhistone H2AX (yH2AX)41. These data suggest that IMMU-132's primarymechanism of action is related to SN-38.

Surface plasmon resonance (BIACORE) analysis did not detect asignificant difference in IMMU-132's binding to the human neonatalreceptor (FcRn), despite the average binding levels being ˜2-fold lowerfor IMMU-132. FcRn binding has been linked to an extended IgG half-lifein serum (Junghans & Anderson, 1996, Proc Natl Acad Sci USA 93:5512-16),but because an antibody's affinity for FcRn in vitro may not correlatewith in vivo clearance rates (Datta-Mannan et al., 2007, J Biol Chem282:1709-17), the overall importance of this finding is unknown.Previous experiments in tumor-bearing mice using ¹¹¹In-DTPA-IMMU-132revealed that the conjugate cleared at a somewhat faster rate from theserum than ¹¹¹In-DTPA-hRS7, although both had similar tumor uptake(Cardillo et al., 2011, Clin Cancer Res 17:3157-69). In the currentstudies, an ELISA assay that also measured the clearance of the IgGcomponent found IMMU-132 and the reduced and NEM-blocked IgG cleared atsimilar rates as unconjugated hRS7, suggesting that the coupling to theinterchain disulfides does not destabilize the antibody. As expected,when using an ELISA that monitored the clearance of the intact conjugate(capturing using an anti-SN-38 antibody and probe with an anti-idiotypeantibody), its clearance rate was faster than when monitoring only theIgG component. This difference simply reflects SN-38's release from theconjugate with a half-life of ˜1 day. We also have examined theclearance rates of hRS7-SN-38 conjugates prepared at differentsubstitution levels by ELISA, and again found no appreciate differencein their clearance rates (Goldenberg et al., 2015, Oncotarget8:22496-512). Overall, these data suggest that mild reduction of theantibody, with the subsequent site-specific modification of some or allinterchain disulfides, has minimal if any impact on the serum clearanceof the IgG, but IMMU-132's overall clearance rate will be definedlargely by the rate of release of SN-38 from the linker.

Additionally, extensive cell-binding experiments demonstrated nosignificant difference in the binding of IMMU-132, the unconjugatedantibody, or the NEM-modified antibody, suggesting that thesite-specific linkage to the interchain disulfides protects theantigen-binding properties of the antibody. Interestingly, when analyzedby BIACORE, which more accurately measures the on-rate and off-rate inaddition to overall affinity, IMMU-132 had a significant 2-foldimprovement in calculated KD-values for Trop-2 binding when compared tonaked hRS7.

We speculate that this improvement may be result of the addedhydrophobicity when SN-38 is conjugated to the antibody. Hydrophobicresidues, as well as hydrophobicity of enclosed regions of proteinbinding sites, have been shown to impart a stronger affinity for theepitope (Park et al., 2000, Nat Biotechnol 18:194-98; Berezov et al.,2001, J Med Chem 44:2565-74; Young et al., 2007, Proc Natl Acad Sci USA104:808013). These regions do not have to be at the protein-proteininterface, but can lie in surrounding, less energetically contactresidues (Li et al., 2005, Structure 13:297-307). While none of theSN-38 conjugation sites are present in the complement-determiningregions (CDR) of hRS7, the prospects that the SN-38 on the antibody maydisplace some of the water molecules around the epitope, resulting inthe improved binding affinity observed for IMMU-132 relative to nakedhRS7, cannot be discounted.

Most efforts in ADC development have been directed towards using astable linker and an ultratoxic drug, with preclinical studiesindicating the specific optimal requirements for those conjugates(Panowski et al., 2014, mAbs 6:34-45; Phillips et al., 2008, Cancer Res68:9280-90). For example, a comparison of T-DM1 to another less stablederivative, T-SSPDM1, revealed that intact T-SSP-DM1 cleared at anapproximately 2-fold faster rate than T-DM-1 in non-tumor-bearing mice(Phillips et al., 2008, Cancer Res 68:9280-90; Erickson et al., 2012,Mol Cancer Ther 11:1133-42), with 1.5-fold higher levels of T-DM1compared to T-SSPDM1 in the tumors. Unexpectedly, and most interesting,was the finding that the amount of free, active maytansinoid catabolitesin the targeted tumors was very similar between the two ADCs (Ericksonet al., 2012, Mol Cancer Ther 11:1133-42).

In other words, T-SSP-DM1 was able to overcome its deficiencies inlinker stability due to the fact that the lower stability resulted inmore efficient release of the drug at the tumor than the more stableT-DM1. Not surprisingly, this equivalency of active drug-catabolitebetween the two ADCs in the tumors resulted in similar anti-tumoreffects in tumor-bearing animals. Ultimately, T-DM1 was chosen based ontoxicity issues that arise when using an ultra-toxic drug and lessstable linkers (Phillips et al., 2008, Cancer Res 68:9280-90). SinceSN-38 is at least a log-fold less toxic than these maytansines, itsrelease from the ADC is expected to have less toxicity. However, evenwith its release in serum, the amount of SN-38 localized in humangastric or pancreatic tumor xenografts was up to 136-fold higher than intumor-bearing mice injected with irinotecan doses that had >20-foldhigher SN-38 equivalents (Sharkey et al., 2015, Clin Cancer Res,21:5131-8). While we have tested more stable linkers in the developmentof IMMU-132, they were significantly less effective in xenograft tumormodels than IMMU-132 (Govindan et al., 2013, Mol Cancer Ther 12:968-78).

Similarly, linkers that released SN-38 more quickly (e.g., serumhalf-life of ˜10 h) also were less effective in xenograft models (Moonet al., 2008, J Med Chem 51:6916-26; Govindan et al., 2009, Clin ChemRes 15:6052-61), suggesting that there is an optimal window at which therelease of SN-38 leads to improved efficacy. Thus, current datademonstrate that IMMU-132 is a more efficient way to target and releasethe drug at the tumor than irinotecan.

Early clinical studies have shown encouraging objective responses invarious solid tumors, and importantly have indicated a better safetyprofile, with a lower incidence of diarrhea, than irinotecan therapy(Starodub et al., 2015, Clin Cancer Res 21:3870-78).

In summary, IMMU-132 (sacituzumab govitecan) is a paradigm-shift in ADCdevelopment. It uses a moderately-stable linker to conjugate 7-8molecules of the more tolerable active metabolite of irinotecan, SN-38,to an anti-Trop-2 antibody. Despite these seemingly counterintuitivecharacteristics vis-a-vis ultra-toxic ADCs, non-clinical studies havedemonstrated that IMMU-132 very effectively targets Trop-2-expressingtumors with significant efficacy and no appreciable toxicity. In earlyphase I/II clinical trials against a wide range of solid tumors,including pancreatic, gastric, TNBC, small-cell and non-small-cell lungcarcinomas, IMMU-132 is likewise exhibiting anti-tumor effects withmanageable toxicities in these patients, with no immune responses toeither the IgG or SN-38 detected, even after many months of dosing(Starodub et al., 2015, Clin Cancer Res 21:3870-78). Given the elevatedexpression of Trop-2 on such a wide variety of solid tumors, IMMU-132continues to be studied clinically, especially in advanced cancers thathave been refractory to most current therapy strategies.

Example 17. Further Results from Phase I/II Clinical Studies

Triple-Negative Breast Cancer (TNBC)

The phase I/II clinical trial (NCT01631552) discussed in the Examplesabove has continued, accruing 56 TNBC patients who were treated with 10mg/kg. The patient population had previously been extensively treatedbefore initiating IMMU-132 therapy, with at least 2 prior lines oftherapy including taxane treatment. Previous treatments includedcyclophosphamide, doxorubicin, carboplatin, gemcitabine, capecitabine,eribulin, cisplatinum, anastrozole, vinorelbine, bevacizumab andtamoxifen. Despite this extensive treatment history TNBC patientsresponded well to IMMU-132, with 2 confirmed complete responses (CR), 13partial responses (PR) and 25 stable disease (SD), for an objectiveresponse rate of 29% (15/52) (FIG. 25). Adding the incidence of CR plusPR plus SD, treatment in TNBC resulted in a a 71% favorable responserate for IMMU-132 treated patients (FIG. 26). The median time toprogression in this heavily pretreated population of TNBC patients was9.4 months, with a range of 2.9 to 14.2 months to date. However, 72% ofpatients in the study were still ongoing treatment. The progression-freesurvival in this group of patients is shown in FIG. 27.

Metastatic NSCLC

The clinical trial is also ongoing for patients with metastaticnon-small cell lung cancer (NSCLC), with 29 assessable patients accruedto date, who were treated with 8 or 10 mg/kg IMMU-132. The bestresponses by RESIST 1.1 criteria are shown in FIG. 28. Out of 29patients, there were 8 PR and 13 SD. The time to progression for NSCLCpatients is shown in FIG. 29, which shows that 21/33 (64%) of NSCLCpatients exhibited PR or SD. The median time to progression was 9/4months, with a range from 1.8 to 15.5+months and 47% of patients stillundergoing treatment. Progression-free survival in NSCLC patientstreated with 8 or 10 mg/kg IMMU-132 is shown in FIG. 30. Median PFS was3.4 months at 8 mg/kg and 3.8 months at 10 mg/kg. However, studies arestill ongoing and the median progression-free survival numbers arelikely to improve.

Metastatic SCLC

Comparable results in metastatic SCLC patients are shown in FIG. 31-33.Best response by RECIST 1.1 for metastatic SCLC patients treated with 8or 10 mg/kg IMMU-132 showed 6 PR and 8 SD out of 25 assessable patients(FIG. 31). Time to progression (FIG. 32) showed a median of 4.9 months,with a range of 1.8 to 15.7+months and 7 patients still undergoingtreatment with IMMU-132. The progression free survival (FIG. 33) showeda median PFS of 2.0 months at 8 mg/kg and 3.6 months at 10 mg/kg. Themedian OS was 8.1 months at 8 mg/kg and could not be determined yet for10 mg/kg.

Urothelial Cancer

Similar results were obtained with urothelial cancer patients treatedwith 8 or 10 mg/kg IMMU-132. The best response daeta for 11 assessablepatients showed 6 PR and 2 SD (FIG. 34). Time to progression (FIG. 35)showed a median of 8.1 months, with a range of 3.6 yo 9.7+months.

In summary, the continuing phase I/II clinical trial shows superiorefficacy of IMMU-132, when administered at the recited dosages of ADC,in at least TNBC, NSCLC, SCLC and urothelial cancers. The superiortherapeutic effect in these heavily pretreated and resistant metastaticcancers occurred without inducing severe toxicities that might precludeclinical use. IMMU-132 showed an acceptable safety profile in heavilypretreated patients with diverse solid cancers, and a median of 2-5prior therapies. Only neutropenia showed an incidence of greater than20% of the patient population for Grade 3 or higher adverse reactions.The study further demonstrates that repeated doses of IMMU-132 may beadministered to human patients, at therapeutic dosages, without evokinginterfering host anti-IMMU-132 antibodies. These results demonstrate thesafety and utility of IMMU-132 for treating diverse Trop-2 positivecancers in human patients.

From the foregoing description, one skilled in the art can easilyascertain the essential characteristics of this invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usage andconditions without undue experimentation. All patents, patentapplications and publications cited herein are incorporated byreference.

We claim:
 1. A method of treating a brain tumor comprising administeringto a human patient with a brain tumor an antibody-drug conjugate (ADC)sacituzumab govitecan; wherein the ADC is administered at a dosage ofbetween 6 mg/kg and 16 mg/kg, wherein the patient has failed to respondto at least one other therapy, prior to treatment with the ADC.
 2. Themethod of claim 1, wherein the dosage is selected from the groupconsisting of 6 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg, 10 mg/kg, 11 mg/kg, 12mg/kg, and 16 mg/kg.
 3. The method of claim 1, wherein the treatmentresults in a reduction in tumor size of at least 15%, at least 20%, atleast 30%, or at least 40%.
 4. The method of claim 1, wherein the braintumor is a metastatic breast cancer, a metastatic prostate cancer, ametastatic lung cancer, a metastatic colon cancer, a metastatic kidneycancer or glioblastoma multiforme.
 5. The method of claim 1, wherein thepatient has relapsed from or failed to respond to treatment withirinotecan, prior to administration of the ADC.
 6. The method of claim1, wherein the tumor is refractory to other therapies but responds tothe ADC.
 7. The method of claim 1, wherein the ADC is administered at adosage of between 8 mg/kg and 12 mg/kg.
 8. The method of claim 1,wherein the ADC is administered at a dosage of between 8 mg/kg and 10mg/kg.
 9. The method of claim 1, further comprising administering to thepatient at least one other anti-cancer therapy selected from the groupconsisting of surgery, external radiation, radioimmunotherapy,immunotherapy, chemotherapy, antisense therapy, interference RNAtherapy, treatment with a therapeutic agent and gene therapy.
 10. Themethod of claim 9, wherein the therapeutic agent is a drug, toxin,immunomodulator, second antibody, antigen-binding fragment of a secondantibody, pro-apoptotic agent, toxin, RNase, hormone, radionuclide,anti-angiogenic agent, siRNA, RNAi, chemotherapeutic agent, cytokine,chemokine, prodrug or enzyme.
 11. The method of claim 10, wherein thedrug is selected from the group consisting of 5-fluorouracil, afatinib,aplidin, azaribine, anastrozole, anthracyclines, axitinib, AVL-101,AVL-291, bendamustine, bleomycin, bortezomib, bosutinib, bryostatin-1,busulfan, calicheamycin, camptothecin, carboplatin,10-hydroxycamptothecin, carmustine, celecoxib, chlorambucil, cisplatin,Cox-2 inhibitors, irinotecan (CPT-11), SN-38, carboplatin, cladribine,camptothecans, crizotinib, cyclophosphamide, cytarabine, dacarbazine,dasatinib, dinaciclib, docetaxel, dactinomycin, daunorubicin,doxorubicin, 2-pyrrolinodoxorubicine (2P-DOX), cyano-morpholinodoxorubicin, doxorubicin glucuronide, epirubicin glucuronide, erlotinib,estramustine, epipodophyllotoxin, erlotinib, entinostat, estrogenreceptor binding agents, etoposide (VP16), etoposide glucuronide,etoposide phosphate, exemestane, fingolimod, floxuridine (FUdR),3′,5′-O-dioleoyl-FudR, fludarabine, flutamide, farnesyl-proteintransferase inhibitors, flavopiridol, fostamatinib, ganetespib,GDC-0834, GS-1101, gefitinib, gemcitabine, hydroxyurea, ibrutinib,idarubicin, idelalisib, ifosfamide, imatinib, L-asparaginase, lapatinib,lenolidamide, leucovorin, LFM-A13, lomustine, mechlorethamine,melphalan, mercaptopurine, 6-mercaptopurine, methotrexate, mitoxantrone,mithramycin, mitomycin, mitotane, navelbine, neratinib, nilotinib,nitrosourea, olaparib, plicomycin, procarbazine, paclitaxel, PCI-32765,pentostatin, PSI-341, raloxifene, semustine, sorafenib, streptozocin,SU11248, sunitinib, tamoxifen, temazolomide, transplatin, thalidomide,thioguanine, thiotepa, teniposide, topotecan, uracil mustard, vatalanib,vinorelbine, vinblastine, vincristine, vinca alkaloids and ZD1839. 12.The method of claim 10, wherein the wherein the immunomodulator isselected from the group consisting of cytokines, lymphokines, monokines,stem cell growth factors, lymphotoxins, hematopoietic factors, colonystimulating factors (CSF), interferons (IFN), parathyroid hormone,thyroxine, insulin, proinsulin, relaxin, prorelaxin, folliclestimulating hormone (FSH), thyroid stimulating hormone (TSH),luteinizing hormone (LH), hepatic growth factor, prostaglandin,fibroblast growth factor, prolactin, placental lactogen, OB protein,transforming growth factor (TGF), TGF-α, TGF-β, insulin-like growthfactor (IGF), erythropoietin, thrombopoietin, tumor necrosis factor(TNF), TNF-α, TNF-β, mullerian-inhibiting substance, mousegonadotropin-associated peptide, inhibin, activin, vascular endothelialgrowth factor, integrin, interleukin (IL), granulocyte-colonystimulating factor (G-CSF), granulocyte macrophage-colony stimulatingfactor (GM-CSF), interferon-α, interferon-β, interferon-γ, S1 factor,IL-1, IL-1 cc, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10,IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18 IL-21, IL-23,IL-25, LIF, kit-ligand, FLT-3, angiostatin, thrombospondin andendostatin.
 13. The method of claim 10, wherein the radionuclide isselected from the group consisting of ¹¹C, ¹³N, ¹⁵O, ³²P, ³³P, ⁴⁷Sc,⁵¹Cr, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, ⁶²Cu, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ⁷⁵Br, ⁷⁵Se, ⁷⁶Br, ⁷⁷As,⁷⁷Br, ^(80m)Br, ⁸⁹Sr, ⁹⁰Y, ⁹⁵Ru, ⁹⁷Ru, ⁹⁹Mo, ^(99m)Tc, ^(103m)Rh, ¹⁰³Ru,¹⁰⁵Rh, ¹⁰⁷Hg, ¹⁰⁹Pd, ¹⁰⁹Pt, ¹¹¹Ag, ¹¹¹In, ^(113m)In, ¹¹⁹Sb, ^(121m)Te,^(122m)Te, ¹²⁵I, ^(125m)Te, ¹²⁶I, ¹³¹I, ¹³³I, ¹⁴²Pr, ¹⁴³Pr, ¹⁴⁹Pm,¹⁵²Dy, ¹⁵³Sm, ¹⁶¹Ho, ¹⁶¹Tb, ¹⁶⁵Tm, ¹⁶⁶Dy, ¹⁶⁶Ho, ¹⁶⁷Tm, ¹⁶⁸Tm, ¹⁶⁹Er,¹⁶⁹Yb, ¹⁷⁷Lu, ¹⁸⁶Re, ¹⁸⁸Re, ^(189m)Os, ¹⁸⁹Re, ¹⁹²Ir, ¹⁹⁴Ir, ¹⁹⁷Pt,¹⁹⁸Au, ¹⁹⁹Au, ²⁰¹Tl, ²⁰³Hg, ²¹¹At, ²¹¹Bi, ²¹¹Pb, ²¹²Bi, ²¹²Pb, ²¹³Bi,²¹⁵Po, ²¹⁷At, ²¹⁹Rn, ²²¹Fr, ²²³Ra, ²²⁵Ac, ²²⁷Th and ²⁵⁵Fm.
 14. Themethod of claim 10, wherein the toxin is selected from the groupconsisting of ricin, abrin, ribonuclease (RNase), DNase I,Staphylococcal enterotoxin-A, pokeweed antiviral protein, gelonin,diphtheria toxin, Pseudomonas exotoxin, and Pseudomonas endotoxin. 15.The method of claim 10, wherein the second antibody is a checkpointinhibitor antibody.
 16. The method of claim 15, wherein the checkpointinhibitor antibody binds to an antigen selected from the groupconsisting of PD-1, PD-L1 and CTLA4.
 17. The method of claim 16, whereinthe checkpoint inhibitor antibody is selected from the group consistingof pembrolizumab, nivolumab, pidilizumab, MDX-1105 (BMS-936559),durvalumab (MEDI4736), atezolizumab (MPDL3280A), ipilimumab andtremelimumab.